Methods, compositions, and kits comprising linker probes for quantifying polynucleotides

ABSTRACT

The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3′ target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3′ target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/490,323, filed Apr. 18, 2017; which is a division of U.S. application Ser. No. 15/009,681, filed Jan. 28, 2016, now U.S. Pat. No. 9,657,346; which is a continuation of U.S. application Ser. No. 13/612,485, filed Sep. 12, 2012, now abandoned; which is a continuation of U.S. application Ser. No. 12/543,466, filed Aug. 18, 2009, now U.S. Pat. No. 9,068,222; which is a continuation of U.S. application Ser. No. 10/947,460, filed Sep. 21, 2004, now U.S. Pat. No. 7,575,863; which claims the benefit of U.S. Provisional Application 60/575,661, filed May 28, 2004, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 6, 2009, is named 533USC1.txt, and is 118,670 bytes in size.

FIELD

The present teachings are in the field of molecular and cell biology, specifically in the field of detecting target polynucleotides such as miRNA.

INTRODUCTION

RNA interference (RNAi) is a highly coordinated, sequence-specific mechanism involved in posttranscriptional gene regulation. During the initial steps of process, a ribonuclease (RNase) II-like enzyme called Dicer reduces long double-strand RNA (dsRNA) and complex hairpin precursors into: 1) small interfering RNAs (siRNA) that degrade messenger RNA (mRNA) and 2) micro RNAs (miRNAs) that can target mRNAs for cleavage or attenuate translation.

The siRNA class of molecules is thought to be comprised of 21-23 nucleotide (nt) duplexes with characteristic dinucleotide 3′ overhangs (Ambros et al., 2003, RNA, 9 (3), 277-279). siRNA has been shown to act as the functional intermediate in RNAi, specifically directing cleavage of complementary mRNA targets in a process that is commonly regarded to be an antiviral cellular defense mechanism (Elbashir et al., 2001, Nature, 411:6836), 494-498, Elbashir et al., 2001, Genes and Development, 15 (2), 188-200). Target RNA cleavage is catalyzed by the RNA-induced silencing complex (RISC), which functions as a siRNA directed endonuclease (reviewed in Bartel, 2004, Cell, 116 (2), 281-297).

Micro RNAs (miRNAs) typically comprise single-stranded, endogenous oligoribonucleotides of roughly 22 (18-25) bases in length that are processed from larger stem-looped precursor RNAs. The first genes recognized to encode miRNAs, lin-4 and let-7 of C. elegans, were identified on the basis of the developmental timing defects associated with the loss-of-function mutations (Lee et al., 1993, Cell, 75 (5), 843-854; Reinhart et al., 2000, Nature, 403, (6772), 901-906; reviewed by Pasquinelli et al., 2002, Annual Review of Cell and Developmental Biology, 18, 495-513). The breadth and importance of miRNA-directed gene regulation are coming into focus as more miRNAs and regulatory targets and functions are discovered. To date, a total of at least 700 miRNAs have been identified in C. elegans, Drosophila (Fire et al., 1998, Nature, 391 (6669), 805-811), mouse, human (Lagos-Quintana et al., 2001, Science, 294 (5543), 853-858), and plants (Reinhart et al., 2002, Genes and Development, 16 (13), 1616-1626). Their sequences are typically conserved among different species. Size ranges from 18 to 25 nucleotides for miRNAs are the most commonly observed to date.

The function of most miRNAs is not known. Recently discovered miRNA functions include control of cell proliferation, cell death, and fat metabolism in flies (Brennecke et al., 2003, cell, 113 (1), 25-36; Xu et al, 2003, Current Biology, 13 (9), 790-795), neuronal patterning in nematodes (Johnston and Hobert, 2003, Nature, 426 (6968), 845-849), modulation of hematopoietic lineage differentiation in mammals (Chen et al., 2004, Science, 303 (5654), 83-87), and control of leaf and flower development in plants (Aukerman and Sakai, 2003, Plant Cell, 15 (11), 2730-2741; Chen, 2003, Science, 303 (5666):2022-2025; Emery et al., 2003, Current Biology, 13 (20), 1768-1774; Palatnik et al., 2003, Nature, 425 (6955), 257-263). There is speculation that miRNAs may represent a new aspect of gene regulation.

Most miRNAs have been discovered by cloning. There are few cloning kits available for researchers from Ambion and QIAGEN etc. The process is laborious and less accurate. Further, there has been little reliable technology available for miRNA quantitation (Allawi et al., Third Wave Technologies, RNA. 2004 July; 10(7):1153-61). Northern blotting has been used but results are not quantitative (Lagos-Quitana et al., 2001, Science, 294 (5543), 853-854). Many miRNA researchers are interested in monitoring the level of the miRNAs at different tissues, at the different stages of development, or after treatment with various chemical agents. However, the short length of miRNAs has their study difficult.

SUMMARY

In some embodiments, the present teachings provide a method for detecting a micro RNA (miRNA) comprising; hybridizing the miRNA and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the miRNA; extending the linker probe to form an extension reaction product; amplifying the extension reaction product to form an amplification product; and, detecting the miRNA.

In some embodiments, the present teachings provide a method for detecting a target polynucleotide comprising; hybridizing the target polynucleotide and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the target polynucleotide; extending the linker probe to form an extension reaction product; amplifying the extension reaction product to form an amplification product in the presence of a detector probe, wherein the detector probe comprises a nucleotide of the linker probe stem in the amplification product or a nucleotide of the linker probe stem complement in the amplification product; and, detecting the target polynucleotide.

In some embodiments, the present teachings provide a method for detecting a miRNA molecule comprising; hybridizing the miRNA molecule and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the target polynucleotide; extending the linker probe to form an extension reaction product; amplifying the extension reaction product in the presence of a detector probe to form an amplification product, wherein the detector probe comprises a nucleotide of the linker probe stem in the amplification product or a nucleotide of the linker probe stem complement in the amplification product, and the detector probe further comprises a nucleotide of the 3′ end region of the miRNA in the amplification product or a nucleotide of the 3′ end region of the miRNA complement in the amplification product; and, detecting the miRNA molecule.

In some embodiments, the present teachings provide a method for detecting two different miRNAs from a single hybridization reaction comprising; hybridizing a first miRNA and a first linker probe, and a second miRNA and a second linker probe, wherein the first linker probe and the second linker probe each comprise a loop, a stem, and a 3′ target-specific portion, wherein the 3′ target-specific portion of the first linker probe base pairs with the 3′ end region of the first miRNA, and wherein the 3′ target-specific portion of the second linker probe base pairs with the 3′ end region of the second miRNA; extending the first linker probe and the second linker probe to form extension reaction products; dividing the extension reaction products into a first amplification reaction to form a first amplification reaction product, and a second amplification reaction to form a second amplification reaction product, wherein a primer in the first amplification reaction corresponds with the first miRNA and not the second miRNA, and a primer in the second amplification reaction corresponds with the second miRNA and not the first miRNA, wherein a first detector probe in the first amplification reaction differs from a second detector probe in the second amplification reaction, wherein the first detector probe comprises a nucleotide of the first linker probe stem of the amplification product or a nucleotide of the first linker probe stem complement in the first amplification product, wherein the second detector probe comprises a nucleotide of the second linker probe stem of the amplification product or a nucleotide of the second linker probe stem complement in the amplification product; and, detecting the two different miRNAs.

In some embodiments, the present teachings provide a method for detecting two different target polynucleotides from a single hybridization reaction comprising; hybridizing a first target polynucleotide and a first linker probe, and a second target polynucleotide and a second linker probe, wherein the first linker probe and the second linker probe each comprise a loop, a stem, and a 3′ target-specific portion, wherein the 3′ target-specific portion of the first linker probe base pairs with the 3′ end region of the first target polynucleotide, and wherein the 3′ target-specific portion of the second linker probe base pairs with the 3′ end region of the second target polynucleotide; extending the first linker probe and the second linker probe to form extension reaction products; dividing the extension reaction products into a first amplification reaction to form a first amplification reaction product and a second amplification reaction to form a second amplification reaction product; and, detecting the two different miRNA molecules.

In some embodiments, the present teachings provide a method for detecting a miRNA molecule from a cell lysate comprising; hybridizing the miRNA molecule from the cell lysate with a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the miRNA; extending the linker probe to form an extension reaction product; amplifying the extension reaction product to form an amplification product in the presence of a detector probe, wherein the detector probe comprises a nucleotide of the linker probe stem of the amplification product or a nucleotide of the linker probe stem complement in the amplification product, and the detector probe further comprises a nucleotide of the 3′ end region of the miRNA in the amplification product or a nucleotide of the 3′ end region of the miRNA complement in the amplification product; and, detecting the miRNA molecule.

A kit comprising; a reverse transcriptase and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion corresponds to a miRNA.

The present teachings contemplate method for detecting a miRNA molecule comprising a step of hybridizing, a step of extending, a step of amplifying, and a step of detecting.

These and other features of the present teachings are set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIGS. 1A, 1B, and 1C depict certain aspects of various compositions according to some embodiments of the present teachings.

FIGS. 2A, 2B, 2C, and 2D depict certain aspects of various compositions according to some embodiments of the present teachings.

FIG. 3 depicts certain sequences of various compositions according to some embodiments of the present teachings. FIG. 3 depicts SEQ ID No. 780, the oligonucleotide for the micro RNA MiR-16 (boxed, 11) and a linker probe (13).

FIG. 4 depicts one single-plex assay design according to some embodiments of the present teachings.

FIG. 5 depicts an overview of a multiplex assay design according to some embodiments of the present teachings.

FIG. 6 depicts a multiplex assay design according to some embodiments of the present teachings.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way. The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, “a primer” means that more than one primer can, but need not, be present; for example but without limitation, one or more copies of a particular primer species, as well as one or more versions of a particular primer type, for example but not limited to, a multiplicity of different forward primers. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.

Some Definitions

As used herein, the term “target polynucleotide” refers to a polynucleotide sequence that is sought to be detected. The target polynucleotide can be obtained from any source, and can comprise any number of different compositional components. For example, the target can be nucleic acid (e.g. DNA or RNA), transfer RNA, siRNA, and can comprise nucleic acid analogs or other nucleic acid mimic. The target can be methylated, non-methylated, or both. The target can be bisulfite-treated and non-methylated cytosines converted to uracil. Further, it will be appreciated that “target polynucleotide” can refer to the target polynucleotide itself, as well as surrogates thereof, for example amplification products, and native sequences. In some embodiments, the target polynucleotide is a miRNA molecule. In some embodiments, the target polynucleotide lacks a poly-A tail. In some embodiments, the target polynucleotide is a short DNA molecule derived from a degraded source, such as can be found in for example but not limited to forensics samples (see for example Butler, 2001, Forensic DNA Typing: Biology and Technology Behind STR Markers. The target polynucleotides of the present teachings can be derived from any of a number of sources, including without limitation, viruses, prokaryotes, eukaryotes, for example but not limited to plants, fungi, and animals. These sources may include, but are not limited to, whole blood, a tissue biopsy, lymph, bone marrow, amniotic fluid, hair, skin, semen, biowarfare agents, anal secretions, vaginal secretions, perspiration, saliva, buccal swabs, various environmental samples (for example, agricultural, water, and soil), research samples generally, purified samples generally, cultured cells, and lysed cells. It will be appreciated that target polynucleotides can be isolated from samples using any of a variety of procedures known in the art, for example the Applied Biosystems ABI Prism™ 6100 Nucleic Acid PrepStation, and the ABI Prism™ 6700 Automated Nucleic Acid Workstation, Boom et al., U.S. Pat. No. 5,234,809, mirVana RNA isolation kit (Ambion), etc. It will be appreciated that target polynucleotides can be cut or sheared prior to analysis, including the use of such procedures as mechanical force, sonication, restriction endonuclease cleavage, or any method known in the art. In general, the target polynucleotides of the present teachings will be single stranded, though in some embodiments the target polynucleotide can be double stranded, and a single strand can result from denaturation.

As used herein, the term “3′ end region of the target polynucleotide” refers to the region of the target to which the 3′ target specific portion of the linker probe hybridizes. In some embodiments there can be a gap between the 3′ end region of the target polynucleotide and the 5′ end of the linker probe, with extension reactions filling in the gap, though generally such scenarios are not preferred because of the likely destabilizing effects on the duplex. In some embodiments, a miRNA molecule is the target, in which case the term “3′ end region of the miRNA” is used.

As used herein, the term “linker probe” refers to a molecule comprising a 3′ target specific portion, a stem, and a loop. Illustrative linker probes are depicted in FIGS. 2A-2D and elsewhere in the present teachings. It will be appreciated that the linker probes, as well as the primers of the present teachings, can be comprised of ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, nucleotide analogs, or combinations thereof. For some illustrative teachings of various nucleotide analogs etc, see Fasman, 1989, Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla., Loakes, N.A.R. 2001, vol 29:2437-2447, and Pellestor et al., Int J Mol Med. 2004 April; 13(4):521-5.), references cited therein, and recent articles citing these reviews. It will be appreciated that the selection of the linker probes to query a given target polynucleotide sequence, and the selection of which collection of target polynucleotide sequences to query in a given reaction with which collection of linker probes, will involve procedures generally known in the art, and can involve the use of algorithms to select for those sequences with minimal secondary and tertiary structure, those targets with minimal sequence redundancy with other regions of the genome, those target regions with desirable thermodynamic characteristics, and other parameters desirable for the context at hand.

As used herein, the term “3′ target-specific portion” refers to the single stranded portion of a linker probe that is complementary to a target polynucleotide. The 3′ target-specific portion is located downstream from the stem of the linker probe. Generally, the 3′ target-specific portion is between 6 and 8 nucleotides long. In some embodiments, the 3′ target-specific portion is 7 nucleotides long. It will be appreciated that routine experimentation can produce other lengths, and that 3′ target-specific portions that are longer than 8 nucleotides or shorter than 6 nucleotides are also contemplated by the present teachings. Generally, the 3′-most nucleotides of the 3′ target-specific portion should have minimal complementarity overlap, or no overlap at all, with the 3′ nucleotides of the forward primer; it will be appreciated that overlap in these regions can produce undesired primer dimer amplification products in subsequent amplification reactions. In some embodiments, the overlap between the 3′-most nucleotides of the 3′ target-specific portion and the 3′ nucleotides of the forward primer is 0, 1, 2, or 3 nucleotides. In some embodiments, greater than 3 nucleotides can be complementary between the 3′-most nucleotides of the 3′ target-specific portion and the 3′ nucleotides of the forward primer, but generally such scenarios will be accompanied by additional non-complementary nucleotides interspersed therein. In some embodiments, modified bases such as LNA can be used in the 3′ target specific portion to increase the Tm of the linker probe (see for example Petersen et al., Trends in Biochemistry (2003), 21:2:74-81). In some embodiments, universal bases can be used, for example to allow for smaller libraries of linker probes. Universal bases can also be used in the 3′ target specific portion to allow for the detection of unknown targets. For some descriptions of universal bases, see for example Loakes et al., Nucleic Acids Research, 2001, Volume 29, No. 12, 2437-2447. In some embodiments, modifications including but not limited to LNAs and universal bases can improve reverse transcription specificity and potentially enhance detection specificity.

As used herein, the term “stem” refers to the double stranded region of the linker probe that is between the 3′ target-specific portion and the loop. Generally, the stem is between 6 and 20 nucleotides long (that is, 6-20 complementary pairs of nucleotides, for a total of 12-40 distinct nucleotides). In some embodiments, the stem is 8-14 nucleotides long. As a general matter, in those embodiments in which a portion of the detector probe is encoded in the stem, the stem can be longer. In those embodiments in which a portion of the detector probe is not encoded in the stem, the stem can be shorter. Those in the art will appreciate that stems shorter that 6 nucleotides and longer than 20 nucleotides can be identified in the course of routine methodology and without undue experimentation, and that such shorter and longer stems are contemplated by the present teachings. In some embodiments, the stem can comprise an identifying portion.

As used herein, the term “loop” refers to a region of the linker probe that is located between the two complementary strands of the stem, as depicted in FIGS. 1A-1C and elsewhere in the present teachings. Typically, the loop comprises single stranded nucleotides, though other moieties modified DNA or RNA, Carbon spacers such as C18, and/or PEG (polyethylene glycol) are also possible. Generally, the loop is between 4 and 20 nucleotides long. In some embodiments, the loop is between 14 and 18 nucleotides long. In some embodiments, the loop is 16 nucleotides long. As a general matter, in those embodiments in which a reverse primer is encoded in the loop, the loop can generally be longer. In those embodiments in which the reverse primer corresponds to both the target polynucleotide as well as the loop, the loop can generally be shorter. Those in the art will appreciate that loops shorter that 4 nucleotides and longer than 20 nucleotides can be identified in the course of routine methodology and without undue experimentation, and that such shorter and longer loops are contemplated by the present teachings. In some embodiments, the loop can comprise an identifying portion.

As used herein, the term “identifying portion” refers to a moiety or moieties that can be used to identify a particular linker probe species, and as a result determine a target polynucleotide sequence, and can refer to a variety of distinguishable moieties including zipcodes, a known number of nucleobases, and combinations thereof. In some embodiments, an identifying portion, or an identifying portion complement, can hybridize to a detector probe, thereby allowing detection of a target polynucleotide sequence in a decoding reaction. The terms “identifying portion complement” typically refers to at least one oligonucleotide that comprises at least one sequence of nucleobases that are at least substantially complementary to and hybridize with their corresponding identifying portion. In some embodiments, identifying portion complements serve as capture moieties for attaching at least one identifier portion:element complex to at least one substrate; serve as “pull-out” sequences for bulk separation procedures; or both as capture moieties and as pull-out sequences (see for example O'Neil, et al., U.S. Pat. Nos. 6,638,760, 6,514,699, 6,146,511, and 6,124,092). Typically, identifying portions and their corresponding identifying portion complements are selected to minimize: internal, self-hybridization; cross-hybridization with different identifying portion species, nucleotide sequences in a reaction composition, including but not limited to gDNA, different species of identifying portion complements, or target-specific portions of probes, and the like; but should be amenable to facile hybridization between the identifying portion and its corresponding identifying portion complement. Identifying portion sequences and identifying portion complement sequences can be selected by any suitable method, for example but not limited to, computer algorithms such as described in PCT Publication Nos. WO 96/12014 and WO 96/41011 and in European Publication No. EP 799,897; and the algorithm and parameters of SantaLucia (Proc. Natl. Acad. Sci. 95:1460-65 (1998)). Descriptions of identifying portions can be found in, among other places, U.S. Pat. No. 6,309,829 (referred to as “tag segment” therein); U.S. Pat. No. 6,451,525 (referred to as “tag segment” therein); U.S. Pat. No. 6,309,829 (referred to as “tag segment” therein); U.S. Pat. No. 5,981,176 (referred to as “grid oligonucleotides” therein); U.S. Pat. No. 5,935,793 (referred to as “identifier tags” therein); and PCT Publication No. WO 01/92579 (referred to as “addressable support-specific sequences” therein). In some embodiments, the stem of the linker probe, the loop of the linker probe, or combinations thereof can comprise an identifying portion, and the detector probe can hybridize to the corresponding identifying portion. In some embodiments, the detector probe can hybridize to both the identifying portion as well as sequence corresponding to the target polynucleotide. In some embodiments, at least two identifying portion: identifying portion complement duplexes have melting temperatures that fall within a Δ T_(m) range (T_(max)−T_(min)) of no more than 10° C. of each other. In some embodiments, at least two identifying portion: identifying portion complement duplexes have melting temperatures that fall within a Δ T_(m) range of 5° C. or less of each other. In some embodiments, at least two identifying portion: identifying portion complement duplexes have melting temperatures that fall within a Δ T_(m) range of 2° C. or less of each other. In some embodiments, at least one identifying portion or at least one identifying portion complement is used to separate the element to which it is bound from at least one component of a ligation reaction composition, a digestion reaction composition, an amplified ligation reaction composition, or the like. In some embodiments, identifying portions are used to attach at least one ligation product, at least one ligation product surrogate, or combinations thereof, to at least one substrate. In some embodiments, at least one ligation product, at least one ligation product surrogate, or combinations thereof, comprise the same identifying portion. Examples of separation approaches include but are not limited to, separating a multiplicity of different element: identifying portion species using the same identifying portion complement, tethering a multiplicity of different element: identifying portion species to a substrate comprising the same identifying portion complement, or both. In some embodiments, at least one identifying portion complement comprises at least one label, at least one mobility modifier, at least one label binding portion, or combinations thereof. In some embodiments, at least one identifying portion complement is annealed to at least one corresponding identifying portion and, subsequently, at least part of that identifying portion complement is released and detected, see for example Published P.C.T. Application WO04/4634 to Rosenblum et al., and Published P.C.T. Application WO01/92579 to Wenz et al.

As used herein, the term “extension reaction” refers to an elongation reaction in which the 3′ target specific portion of a linker probe is extended to form an extension reaction product comprising a strand complementary to the target polynucleotide. In some embodiments, the target polynucleotide is a miRNA molecule and the extension reaction is a reverse transcription reaction comprising a reverse transcriptase. In some embodiments, the extension reaction is a reverse transcription reaction comprising a polymerase derived from a Eubacteria. In some embodiments, the extension reaction can comprise rTth polymerase, for example as commercially available from Applied Biosystems catalog number N808-0192, and N808-0098. In some embodiments, the target polynucleotide is a miRNA or other RNA molecule, and as such it will be appreciated that the use of polymerases that also comprise reverse transcription properties can allow for some embodiments of the present teachings to comprise a first reverse transcription reaction followed thereafter by an amplification reaction, thereby allowing for the consolidation of two reactions in essentially a single reaction. In some embodiments, the target polynucleotide is a short DNA molecule and the extension reaction comprises a polymerase and results in the synthesis of a 2^(nd) strand of DNA. In some embodiments, the consolidation of the extension reaction and a subsequent amplification reaction is further contemplated by the present teachings.

As used herein, the term “primer portion” refers to a region of a polynucleotide sequence that can serve directly, or by virtue of its complement, as the template upon which a primer can anneal for any of a variety of primer nucleotide extension reactions known in the art (for example, PCR). It will be appreciated by those of skill in the art that when two primer portions are present on a single polynucleotide, the orientation of the two primer portions is generally different. For example, one PCR primer can directly hybridize to a first primer portion, while the other PCR primer can hybridize to the complement of the second primer portion. In addition, “universal” primers and primer portions as used herein are generally chosen to be as unique as possible given the particular assays and host genomes to ensure specificity of the assay.

As used herein, the term “forward primer” refers to a primer that comprises an extension reaction product portion and a tail portion. The extension reaction product portion of the forward primer hybridizes to the extension reaction product. Generally, the extension reaction product portion of the forward primer is between 9 and 19 nucleotides in length. In some embodiments, the extension reaction product portion of the forward primer is 16 nucleotides. The tail portion is located upstream from the extension reaction product portion, and is not complementary with the extension reaction product; after a round of amplification however, the tail portion can hybridize to complementary sequence of amplification products. Generally, the tail portion of the forward primer is between 5-8 nucleotides long. In some embodiments, the tail portion of the forward primer is 6 nucleotides long. Those in the art will appreciate that forward primer tail portion lengths shorter than 5 nucleotides and longer than 8 nucleotides can be identified in the course of routine methodology and without undue experimentation, and that such shorter and longer forward primer tail portion lengths are contemplated by the present teachings. Further, those in the art will appreciate that lengths of the extension reaction product portion of the forward primer shorter than 9 nucleotides in length and longer than 19 nucleotides in length can be identified in the course of routine methodology and without undue experimentation, and that such shorter and longer extension reaction product portion of forward primers are contemplated by the present teachings.

As used herein, the term “reverse primer” refers to a primer that when extended forms a strand complementary to the target polynucleotide. In some embodiments, the reverse primer corresponds with a region of the loop of the linker probe. Following the extension reaction, the forward primer can be extended to form a second strand product. The reverse primer hybridizes with this second strand product, and can be extended to continue the amplification reaction. In some embodiments, the reverse primer corresponds with a region of the loop of the linker probe, a region of the stem of the linker probe, a region of the target polynucleotide, or combinations thereof. Generally, the reverse primer is between 13-16 nucleotides long. In some embodiments the reverse primer is 14 nucleotides long. In some embodiments, the reverse primer can further comprise a non-complementary tail region, though such a tail is not required. In some embodiments, the reverse primer is a “universal reverse primer,” which indicates that the sequence of the reverse primer can be used in a plurality of different reactions querying different target polynucleotides, but that the reverse primer nonetheless is the same sequence.

The term “upstream” as used herein takes on its customary meaning in molecular biology, and refers to the location of a region of a polynucleotide that is on the 5′ side of a “downstream” region. Correspondingly, the term “downstream” refers to the location of a region of a polynucleotide that is on the 3′ side of an “upstream” region.

As used herein, the term “hybridization” refers to the complementary base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex, triplex, or other higher-ordered structure, and is used herein interchangeably with “annealing.” Typically, the primary interaction is base specific, e.g., A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding. Base-stacking and hydrophobic interactions can also contribute to duplex stability. Conditions for hybridizing detector probes and primers to complementary and substantially complementary target sequences are well known, e.g., as described in Nucleic Acid Hybridization, A Practical Approach, B. Hames and S. Higgins, eds., IRL Press, Washington, D.C. (1985) and J. Wetmur and N. Davidson, Mol. Biol. 31:349 et seq. (1968). In general, whether such annealing takes place is influenced by, among other things, the length of the polynucleotides and the complementary, the pH, the temperature, the presence of mono- and divalent cations, the proportion of G and C nucleotides in the hybridizing region, the viscosity of the medium, and the presence of denaturants. Such variables influence the time required for hybridization. Thus, the preferred annealing conditions will depend upon the particular application. Such conditions, however, can be routinely determined by the person of ordinary skill in the art without undue experimentation. It will be appreciated that complementarity need not be perfect; there can be a small number of base pair mismatches that will minimally interfere with hybridization between the target sequence and the single stranded nucleic acids of the present teachings. However, if the number of base pair mismatches is so great that no hybridization can occur under minimally stringent conditions then the sequence is generally not a complementary target sequence. Thus, complementarity herein is meant that the probes or primers are sufficiently complementary to the target sequence to hybridize under the selected reaction conditions to achieve the ends of the present teachings.

As used herein, the term “amplifying” refers to any means by which at least a part of a target polynucleotide, target polynucleotide surrogate, or combinations thereof, is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Exemplary means for performing an amplifying step include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA) and the like, including multiplex versions or combinations thereof, for example but not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known as combined chain reaction—CCR), and the like. Descriptions of such techniques can be found in, among other places, Sambrook et al. Molecular Cloning, 3^(rd) Edition; Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002), Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, NJ (2002); Abramson et al., Curr Opin Biotechnol. 1993 February; 4(1):41-7, U.S. Pat. Nos. 6,027,998; 6,605,451, Barany et al., PCT Publication No. WO 97/31256; Wenz et al., PCT Publication No. WO 01/92579; Day et al., Genomics, 29(1): 152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the world wide web at: promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 November; 2(6):542-8, Cook et al., J Microbiol Methods. 2003 May; 53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February; 12(1):21-7, U.S. Pat. Nos. 5,830,711, 6,027,889, 5,686,243, Published P.C.T. Application WO0056927A3, and Published P.C.T. Application WO9803673A1. In some embodiments, newly-formed nucleic acid duplexes are not initially denatured, but are used in their double-stranded form in one or more subsequent steps. An extension reaction is an amplifying technique that comprises elongating a linker probe that is annealed to a template in the 5′ to 3′ direction using an amplifying means such as a polymerase and/or reverse transcriptase. According to some embodiments, with appropriate buffers, salts, pH, temperature, and nucleotide triphosphates, including analogs thereof, i.e., under appropriate conditions, a polymerase incorporates nucleotides complementary to the template strand starting at the 3′-end of an annealed linker probe, to generate a complementary strand. In some embodiments, the polymerase used for extension lacks or substantially lacks 5′ exonuclease activity. In some embodiments of the present teachings, unconventional nucleotide bases can be introduced into the amplification reaction products and the products treated by enzymatic (e.g., glycosylases) and/or physical-chemical means in order to render the product incapable of acting as a template for subsequent amplifications. In some embodiments, uracil can be included as a nucleobase in the reaction mixture, thereby allowing for subsequent reactions to decontaminate carryover of previous uracil-containing products by the use of uracil-N-glycosylase (see for example Published P.C.T. Application WO9201814A2). In some embodiments of the present teachings, any of a variety of techniques can be employed prior to amplification in order to facilitate amplification success, as described for example in Radstrom et al., Mol Biotechnol. 2004 February; 26(2):133-46. In some embodiments, amplification can be achieved in a self-contained integrated approach comprising sample preparation and detection, as described for example in U.S. Pat. Nos. 6,153,425 and 6,649,378. Reversibly modified enzymes, for example but not limited to those described in U.S. Pat. No. 5,773,258, are also within the scope of the disclosed teachings. The present teachings also contemplate various uracil-based decontamination strategies, wherein for example uracil can be incorporated into an amplification reaction, and subsequent carry-over products removed with various glycosylase treatments (see for example U.S. Pat. No. 5,536,649, and U.S. Provisional Application 60/584,682 to Andersen et al.). Those in the art will understand that any protein with the desired enzymatic activity can be used in the disclosed methods and kits. Descriptions of DNA polymerases, including reverse transcriptases, uracil N-glycosylase, and the like, can be found in, among other places, Twyman, Advanced Molecular Biology, BIOS Scientific Publishers, 1999; Enzyme Resource Guide, rev. 092298, Promega, 1998; Sambrook and Russell; Sambrook et al.; Lehninger; PCR: The Basics; and Ausbel et al.

As used herein, the term “detector probe” refers to a molecule used in an amplification reaction, typically for quantitative or real-time PCR analysis, as well as end-point analysis. Such detector probes can be used to monitor the amplification of the target polynucleotide. In some embodiments, detector probes present in an amplification reaction are suitable for monitoring the amount of amplicon(s) produced as a function of time. Such detector probes include, but are not limited to, the 5′-exonuclease assay (TaqMan® probes described herein (see also U.S. Pat. No. 5,538,848) various stem-loop molecular beacons (see e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, Nature Biotechnology 14:303-308), stemless or linear beacons (see, e.g., WO 99/21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097), Sunrise®/Amplifluor® probes (U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion™ probes (Solinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743), bulge loop probes (U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons (U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al., 1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, Molecular Cell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35; Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002, Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, Nucleic Acids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332; Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al., 2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem Res. Toxicol. 15:118-126; and Yu et al., 2001, J. Am. Chem. Soc 14:11155-11161. Detector probes can also comprise quenchers, including without limitation black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch). Detector probes can also comprise two probes, wherein for example a fluor is on one probe, and a quencher is on the other probe, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization on the target alters the signal signature via a change in fluorescence. Detector probes can also comprise sulfonate derivatives of fluorescenin dyes with SO3 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY 5 (commercially available for example from Amersham). In some embodiments, interchelating labels are used such as ethidium bromide, SYBR® Green I (Molecular Probes), and PicoGreen® (Molecular Probes), thereby allowing visualization in real-time, or end point, of an amplification product in the absence of a detector probe. In some embodiments, real-time visualization can comprise both an intercalating detector probe and a sequence-based detector probe can be employed. In some embodiments, the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction, and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction. In some embodiments, the detector probes of the present teachings have a Tm of 63-69 C, though it will be appreciated that guided by the present teachings routine experimentation can result in detector probes with other Tms. In some embodiments, probes can further comprise various modifications such as a minor groove binder (see for example U.S. Pat. No. 6,486,308) to further provide desirable thermodynamic characteristics. In some embodiments, detector probes can correspond to identifying portions or identifying portion complements.

The term “corresponding” as used herein refers to a specific relationship between the elements to which the term refers. Some non-limiting examples of corresponding include: a linker probe can correspond with a target polynucleotide, and vice versa. A forward primer can correspond with a target polynucleotide, and vice versa. A linker probe can correspond with a forward primer for a given target polynucleotide, and vice versa. The 3′ target-specific portion of the linker probe can correspond with the 3′ region of a target polynucleotide, and vice versa. A detector probe can correspond with a particular region of a target polynucleotide and vice versa. A detector probe can correspond with a particular identifying portion and vice versa. In some cases, the corresponding elements can be complementary. In some cases, the corresponding elements are not complementary to each other, but one element can be complementary to the complement of another element.

The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

As used herein, the term “reaction vessel” generally refers to any container in which a reaction can occur in accordance with the present teachings. In some embodiments, a reaction vessel can be an eppendorf tube, and other containers of the sort in common practice in modern molecular biology laboratories. In some embodiments, a reaction vessel can be a well in microtitre plate, a spot on a glass slide, or a well in an Applied Biosystems TaqMan Low Density Array for gene expression (formerly MicroCard™). For example, a plurality of reaction vessels can reside on the same support. In some embodiments, lab-on-a-chip like devices, available for example from Caliper and Fluidgm, can provide for reaction vessels. In some embodiments, various microfluidic approaches as described in U.S. Provisional Application 60/545,674 to Wenz et al., can be employed. It will be recognized that a variety of reaction vessel are available in the art and within the scope of the present teachings.

As used herein, the term “detection” refers to any of a variety of ways of determining the presence and/or quantity and/or identity of a target polynucleoteide. In some embodiments employing a donor moiety and signal moiety, one may use certain energy-transfer fluorescent dyes. Certain nonlimiting exemplary pairs of donors (donor moieties) and acceptors (signal moieties) are illustrated, e.g., in U.S. Pat. Nos. 5,863,727; 5,800,996; and 5,945,526. Use of some combinations of a donor and an acceptor have been called FRET (Fluorescent Resonance Energy Transfer). In some embodiments, fluorophores that can be used as signaling probes include, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5 (Cy 5), fluorescein, Vic™, Liz™, Tamra™, 5-Fam™, 6-Fam™, and Texas Red (Molecular Probes). (Vic™, Liz™, Tamra™, 5-Fam™, and 6-Fam™ (all available from Applied Biosystems, Foster City, CA.). In some embodiments, the amount of detector probe that gives a fluorescent signal in response to an excited light typically relates to the amount of nucleic acid produced in the amplification reaction. Thus, in some embodiments, the amount of fluorescent signal is related to the amount of product created in the amplification reaction. In such embodiments, one can therefore measure the amount of amplification product by measuring the intensity of the fluorescent signal from the fluorescent indicator. According to some embodiments, one can employ an internal standard to quantify the amplification product indicated by the fluorescent signal. See, e.g., U.S. Pat. No. 5,736,333. Devices have been developed that can perform a thermal cycling reaction with compositions containing a fluorescent indicator, emit a light beam of a specified wavelength, read the intensity of the fluorescent dye, and display the intensity of fluorescence after each cycle. Devices comprising a thermal cycler, light beam emitter, and a fluorescent signal detector, have been described, e.g., in U.S. Pat. Nos. 5,928,907; 6,015,674; and 6,174,670, and include, but are not limited to the ABI Prism® 7700 Sequence Detection System (Applied Biosystems, Foster City, California), the ABI GeneAmp® 5700 Sequence Detection System (Applied Biosystems, Foster City, California), the ABI GeneAmp® 7300 Sequence Detection System (Applied Biosystems, Foster City, California), and the ABI GeneAmp® 7500 Sequence Detection System (Applied Biosystems). In some embodiments, each of these functions can be performed by separate devices. For example, if one employs a Q-beta replicase reaction for amplification, the reaction may not take place in a thermal cycler, but could include a light beam emitted at a specific wavelength, detection of the fluorescent signal, and calculation and display of the amount of amplification product. In some embodiments, combined thermal cycling and fluorescence detecting devices can be used for precise quantification of target nucleic acid sequences in samples. In some embodiments, fluorescent signals can be detected and displayed during and/or after one or more thermal cycles, thus permitting monitoring of amplification products as the reactions occur in “real time.” In some embodiments, one can use the amount of amplification product and number of amplification cycles to calculate how much of the target nucleic acid sequence was in the sample prior to amplification. In some embodiments, one could simply monitor the amount of amplification product after a predetermined number of cycles sufficient to indicate the presence of the target nucleic acid sequence in the sample. One skilled in the art can easily determine, for any given sample type, primer sequence, and reaction condition, how many cycles are sufficient to determine the presence of a given target polynucleotide. As used herein, determining the presence of a target can comprise identifying it, as well as optionally quantifying it. In some embodiments, the amplification products can be scored as positive or negative as soon as a given number of cycles is complete. In some embodiments, the results may be transmitted electronically directly to a database and tabulated. Thus, in some embodiments, large numbers of samples can be processed and analyzed with less time and labor when such an instrument is used. In some embodiments, different detector probes may distinguish between different target polynucleoteides. A non-limiting example of such a probe is a 5′-nuclease fluorescent probe, such as a TaqMan® probe molecule, wherein a fluorescent molecule is attached to a fluorescence-quenching molecule through an oligonucleotide link element. In some embodiments, the oligonucleotide link element of the 5′-nuclease fluorescent probe binds to a specific sequence of an identifying portion or its complement. In some embodiments, different 5′-nuclease fluorescent probes, each fluorescing at different wavelengths, can distinguish between different amplification products within the same amplification reaction. For example, in some embodiments, one could use two different 5′-nuclease fluorescent probes that fluoresce at two different wavelengths (WL_(A) and WL_(B)) and that are specific to two different stem regions of two different extension reaction products (A′ and B′, respectively). Amplification product A′ is formed if target nucleic acid sequence A is in the sample, and amplification product B′ is formed if target nucleic acid sequence B is in the sample. In some embodiments, amplification product A′ and/or B′ may form even if the appropriate target nucleic acid sequence is not in the sample, but such occurs to a measurably lesser extent than when the appropriate target nucleic acid sequence is in the sample. After amplification, one can determine which specific target nucleic acid sequences are present in the sample based on the wavelength of signal detected and their intensity. Thus, if an appropriate detectable signal value of only wavelength WL_(A) is detected, one would know that the sample includes target nucleic acid sequence A, but not target nucleic acid sequence B. If an appropriate detectable signal value of both wavelengths WL_(A) and WL_(B) are detected, one would know that the sample includes both target nucleic acid sequence A and target nucleic acid sequence B. In some embodiments, detection can occur through any of a variety of mobility dependent analytical techniques based on differential rates of migration between different analyte species. Exemplary mobility-dependent analysis techniques include electrophoresis, chromatography, mass spectroscopy, sedimentation, e.g., gradient centrifugation, field-flow fractionation, multi-stage extraction techniques, and the like. In some embodiments, mobility probes can be hybridized to amplification products, and the identity of the target polynucleotide determined via a mobility dependent analysis technique of the eluted mobility probes, as described for example in Published P.C.T. Application WO04/46344 to Rosenblum et al., and WO01/92579 to Wenz et al. In some embodiments, detection can be achieved by various microarrays and related software such as the Applied Biosystems Array System with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and other commercially available array systems available from Affymetrix, Agilent, Illumina, and Amersham Biosciences, among others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; and Stears et al., Nat. Med. 9:140-45, including supplements, 2003). It will also be appreciated that detection can comprise reporter groups that are incorporated into the reaction products, either as part of labeled primers or due to the incorporation of labeled dNTPs during an amplification, or attached to reaction products, for example but not limited to, via hybridization tag complements comprising reporter groups or via linker arms that are integral or attached to reaction products. Detection of unlabeled reaction products, for example using mass spectrometry, is also within the scope of the current teachings.

Exemplary Embodiments

FIGS. 1A-1C depict certain compositions according to some embodiments of the present teachings. FIG. 1A, a miRNA molecule (1, dashed line) is depicted. FIG. 1B, a linker probe (2) is depicted, illustrating a 3′ target specific portion (3), a stem (4), and a loop (5). FIG. 1C, a miRNA hybridized to a linker probe is depicted, illustrating the 3′ target specific portion of the linker probe (3) hybridized to the 3′ end region of the miRNA (6).

As shown in FIGS. 2A-2D, a target polynucleotide (9, dotted line) is illustrated to show the relationship with various components of the linker probe (10), the detector probe (7), and the reverse primer (8), according to various non-limiting embodiments of the present teachings. For example as shown in FIG. 2A, in some embodiments the detector probe (7) can correspond with the 3′ end region of the target polynucleotide in the amplification product as well as a region upstream from the 3′ end region of the target polynucleotide in the amplification product. (Here, the detector probe is depicted as rectangle (7) with an F and a Q, symbolizing a TaqMan probe with a florophore (F) and a quencher (Q)). Also shown in FIG. 2A, the loop can correspond to the reverse primer (8). In some embodiments as shown in FIG. 2B, the detector probe (7) can correspond with a region of the amplification product corresponding with the 3′ end region of the target polynucleotide in the amplification product, as well as a region upstream from the 3′ end region of the target polynucleotide in the amplification product, as well as the linker probe stem in the amplification product. Also shown in FIG. 2B, the upstream region of the stem, as well as the loop, can correspond to the reverse primer (8). In some embodiments as shown in FIG. 2C, the detector probe can correspond to the amplification product in a manner similar to that shown in FIG. 2B, but the loop can correspond to the reverse primer (8). In some embodiments as shown in FIG. 2D, the detector probe (7) can correspond with the linker probe stem in the amplification product. Also shown in FIG. 2D, the upstream region of the stem, as well as the loop can correspond to the reverse primer (8). It will be appreciated that various related strategies for implementing the different functional regions of these compositions are possible in light of the present teachings, and that such derivations are routine to one having ordinary skill in the art without undue experimentation.

FIG. 3 depicts the nucleotide relationship for the micro RNA MiR-16 (boxed, 11) according to some embodiments of the present teachings. Shown here is the interrelationship of MiR-16 to a forward primer (12) (SEQ ID No. 781), a linker probe (13), a TaqMan detector probe (14) (SEQ ID No. 782), and a reverse primer (boxed, 15) (SEQ ID No. 783). The TaqMan probe comprises a 3′ minor groove binder (MGB), and a 5′ FAM florophore. It will be appreciated that in some embodiments of the present teachings the detector probes, such as for example TaqMan probes, can hybridize to either strand of an amplification product. For example, in some embodiments the detector probe can hybridize to the strand of the amplification product corresponding to the first strand synthesized. In some embodiments, the detector probe can hybridize to the strand of the amplification product corresponding to the second strand synthesized.

FIG. 4 depicts a single-plex assay design according to some embodiments of the present teachings. Here, a miRNA molecule (16) and a linker probe (17) are hybridized together (18). The 3′ end of the linker probe of the target-linker probe composition is extended to form an extension product (19) that can be amplified in a PCR. The PCR can comprise a miRNA specific forward primer (20) and a reverse primer (21). The detection of a detector probe (22) during the amplification allows for quantitation of the miRNA.

FIG. 5 depicts an overview of a multiplex assay design according to some embodiments of the present teachings. Here, a multiplexed hybridization and extension reaction is performed in a first reaction vessel (23). Thereafter, aliquots of the extension reaction products from the first reaction vessel are transferred into a plurality of amplification reactions (here, depicted as PCRs 1, 2, and 3) in a plurality of second reaction vessels. Each PCR can comprise a distinct primer pair and a distinct detector probe. In some embodiments, a distinct primer pair but the same detector probe can be present in each of a plurality of PCRs.

FIG. 6 depicts a multiplex assay design according to some embodiments of the present teachings. Here, three different miRNAs (24, 25, and 26) are queried in a hybridization reaction comprising three different linker probes (27, 28, and 29). Following hybridization and extension to form extension products (30, 31, and 32), the extension products are divided into three separate amplification reactions. (Though not explicitly shown, it will be appreciated that a number of copies of the molecules depicted by 30, 31, and 32 can be present, such that each of the three amplification reactions can have copies of 30, 31, and 32.) PCR 1 comprises a forward primer specific for miRNA 24 (33), PCR 2 comprises a forward primer specific for miRNA 25 (34), and PCR 3 comprises a forward primer specific for miRNA 26 (35). Each of the forward primers further comprise a non-complementary tail portion. PCR 1, PCR 2, and PCR 3 all comprise the same universal reverse primer 36. Further, PCR 1 comprises a distinct detector probe (37) that corresponds to the 3′ end region of miRNA 24 and the stem of linker probe 27, PCR 2 comprises a distinct detector probe (38) that corresponds to the 3′ end region of miRNA 25 and the stem of linker probe 28, and PCR 3 comprises a distinct detector probe (39) that corresponds to the 3′ region of miRNA 26 and the stem of linker probe 29.

The present teachings also contemplate reactions comprising configurations other than a linker probe. For example, in some embodiments, two hybridized molecules with a sticky end can be employed, wherein for example an overlapping 3′ sticky end hybridizes with the 3′ end region of the target polynucleotide. Some descriptions of two molecule configurations that can be employed in the present teachings can be found in Chen et al., U.S. Provisional Application 60/517,470. Viewed in light of the present teachings herein, one of skill in the art will appreciate that the approaches of Chen et al., can also be employed to result in extension reaction products that are longer that the target polynucleotide. These longer products can be detected with detector probes by, for example, taking advantage of the additional nucleotides introduced into the reaction products.

The present teachings also contemplate embodiments wherein the linker probe is ligated to the target polynucleotide, as described for example in Chen et al., U.S. Provisional Application 60/575,661, and the corresponding co-filed U.S. Provisional application co-filed herewith

Further, it will be appreciated that in some embodiments of the present teachings, the two molecule configurations in Chen et al., U.S. Provisional Application 60/517,470 can be applied in embodiments comprising the linker approaches discussed in Chen et al., U.S. Provisional Application 60/575,661.

Generally however, the loop structure of the present teachings will enhance the Tm of the target polynucleotide-linker probe duplex. Without being limited to any particular theory, this enhanced Tm could possibly be due to base stacking effects. Also, the characteristics of the looped linker probe of the present teachings can minimize nonspecific priming during the extension reaction, and/or a subsequent amplification reaction such as PCR. Further, the looped linker probe of the present teachings can better differentiate mature and precursor forms of miRNA, as illustrated infra in Example 6.

The present teachings also contemplate encoding and decoding reaction schemes, wherein a first encoding extension reaction is followed by a second decoding amplification reaction, as described for example in Livak et al., U.S. Provisional Application 60/556,162, Chen et al., U.S. Provisional Application 60/556,157, Andersen et al., U.S. Provisional Application 60/556,224, and Lao et al., U.S. Provisional Application 60/556,163.

The present teachings also contemplate a variety of strategies to minimize the number of different molecules in multiplexed amplification strategies, as described for example in Whitcombe et al., U.S. Pat. No. 6,270,967.

In certain embodiments, the present teachings also provide kits designed to expedite performing certain methods. In some embodiments, kits serve to expedite the performance of the methods of interest by assembling two or more components used in carrying out the methods. In some embodiments, kits may contain components in pre-measured unit amounts to minimize the need for measurements by end-users. In some embodiments, kits may include instructions for performing one or more methods of the present teachings. In certain embodiments, the kit components are optimized to operate in conjunction with one another.

For example, the present teachings provide a kit comprising, a reverse transcriptase and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion corresponds to a miRNA. In some embodiments, the kits can comprise a DNA polymerase. In some embodiments, the kits can comprise a primer pair. In some embodiments, the kits can further comprise a forward primer specific for a miRNA, and, a universal reverse primer, wherein the universal reverse primer comprises a nucleotide of the loop of the linker probe. In some embodiments, the kits can comprise a plurality of primer pairs, wherein each primer pair is in one reaction vessel of a plurality of reaction vessels. In some embodiments, the kits can comprise a detector probe. In some embodiments, the detector probe comprises a nucleotide of the linker probe stem in the amplification product or a nucleotide of the linker probe stem complement in the amplification product, and the detector probe further comprises a nucleotide of the 3′ end region of the miRNA in the amplification product or a nucleotide of the 3′ end region of the miRNA complement in the amplification product.

The present teachings further contemplate kits comprising a means for hybridizing, a means for extending, a means for amplifying, a means for detecting, or combinations thereof.

While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings. Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the teachings in any way.

Example 1

A single-plex reaction was performed in replicate for a collection of mouse miRNAs, and the effect of the presence or absence of ligase, as well as the presence or absence of reverse transcriptase, determined. The results are shown in Table 1 as Ct values.

First, a 6 ul reaction was set up comprising: 1 ul Reverse Transcription Enzyme Mix (Applied Biosystems part number 4340444) (or 1 ul dH2O), 0.5 ul T4 DNA Ligase (400 units/ul, NEB) (or 0.5 ul dH20), 0.25 ul 2M KCl, 0.05 ul dNTPs (25 mM each), 0.25 ul T4 Kinase (10 units/ul, NEB), 1 ul 10× T4 DNA ligase buffer (NEB), 0.25 ul Applied Biosystems RNase Inhibitor (10 units/ul), and 2.2 ul dH20 Next, 2 ul of the linker probe (0.25 uM) and RNA samples (2 ul of 0.25 ug/ul mouse lung total RNA (Ambion, product number 7818) were added. Next, the reaction was mixed, spun briefly, and placed on ice for 5 minutes.

The reaction was then incubated at 16 C for 30 minutes, 42 C for 30 minutes, followed by 85 C for 5 minutes, and then held at 4 C. The reactions were diluted 4 times by adding 30 ul of dH20 prior to the PCR amplification.

A 10 ul PCR amplification was then set up comprising: 2 ul of diluted reverse transcription reaction product, 1.3 ul 10 uM miRNA specific Forward Primer, 0.7 ul 10 uM Universal Reverse Primer, 0.2 ul TaqMan detector probe, 0.2 ul dNTPs (25 mM each), 0.6 ul dH20, 5 ul 2× TaqMan master mix (Applied Biosystems, without UNG).The reaction was started with a 95 C step for 10 minutes. Then, 40 cycles were performed, each cycle comprising 95 C for 15 seconds, and 60 C for 1 minute. Table 1 indicates the results of this experiment.

TABLE 1 Reverse miRNA Replicate Ligase transcriptase Let-7a1 mir16 mir20 mir21 mir26a mir30a mir224 average Yes Yes 16.8 16.0 19.1 16.8 15.0 21.3 27.3 18.9 Yes No 38.7 31.3 39.9 31.9 30.1 33.3 40.0 35.0 I No Yes 18.0 14.6 18.3 16.2 14.0 21.3 26.4 18.4 No No 40.0 36.6 40.0 40.0 33.8 39.2 40.0 38.5 Yes Yes 17.1 16.2 19.3 17.0 15.1 21.4 27.3 19.1 Yes No 38.9 31.2 37.6 32.1 30.4 33.4 39.4 34.7 II No Yes 18.4 14.8 18.7 16.6 14.3 21.5 26.7 18.7 No No 40.0 36.1 40.0 40.0 34.1 40.0 40.0 38.6 Replicate Yes Yes 16.9 16.1 19.2 16.9 15.0 21.4 27.3 19.0 Average Yes No 38.8 31.2 38.8 32.0 30.3 33.4 39.7 34.9 No Yes 18.2 14.7 18.5 16.4 14.1 21.4 26.6 18.6 No No 40.0 36.4 40.0 40.0 34.0 39.6 40.0 40.0

Sequences of corresponding forward primers, reverse primer, and TaqMan probes are shown in Table 2.

TABLE 2 SEQ ID miRNA ID NO: miRNA sequences miR-16 1 uagcagcacguaaauauuggcg miR-20 2 uaaagugcuuauagugcaggua miR-21 3 uagcuuaucagacugauguuga miR-22 4 aagcugccaguugaagaacugu miR-26a 5 uucaaguaauccaggauaggcu miR-29 6 cuagcaccaucugaaaucgguu miR-30a 7 cuuucagucggauguuugcagc miR-34 8 uggcagugucuuagcugguugu miR-200b 9 cucuaauacugccugguaaugaug miR-323 10 gcacauuacacggucgaccucu miR-324-5 11 cgcauccccuagggcauuggugu let-7a1 12 ugagguaguagguuguauaguu SEQ ID Linker probe NO: Linker probe sequences miR-16linR6 13 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC CGCCAA miR20LinR6 14 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC TACCTG miR-21linR6 15 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC TCAACA miR-22linR6 16 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC ACAGTT miR-26alinR6 17 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC AGCCTA miR-29linR6 18 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC AACCGA miR30LinR6 19 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC GCTGCA miR-34linR6 20 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC ACAACC miR-200blinR6 21 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC CATCAT miR-323linR6 22 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC AGAGGT miR-324-5linR6 23 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC ACACCA let7aLinR6 24 GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC AACTAT Forward  SEQ ID primer ID NO: Forward primer sequences miR-16F55 25 CGCGCTAGCAGCACGTAAAT miR-20F56 26 GCCGCTAAAGTGCTTATAGTGC miR-21F56 27 GCCCGCTAGCTTATCAGACTGATG miR-22F56 28 GCCTGAAGCTGCCAGTTGA miR-26aF54 29 CCGGCGTTCAAGTAATCCAGGA miR-29F56 30 GCCGCTAGCACCATCTGAAA miR-30aF58 31 GCCCCTTTCAGTCGGATGTTT miR-34F56 32 GCCCGTGGCAGTGTCTTAG miR-200bF56 33 GCCCCTCTAATACTGCCTGG miR-323F58 34 GCCACGCACATTACACGGTC miR-324-5F56 35 GCCACCATCCCCTAGGGC let-7a1F56 36 GCCGCTGAGGTAGTAGGTTGT TaqMan  SEQ ID probe ID NO: TaqMan probe sequences miR-16_Tq8F67 37 (6FAM)ATACGACCGCCAATAT(MGB) miR20_Tq8F68 38 (6FAM)CTGGATACGACTACCTG(MGB) miR-21_Tq8F68 39 (6FAM)CTGGATACGACTCAACA(MGB) miR-22_Tq8F68 40 (6FAM)TGGATACGACACAGTTCT(MGB) miR-26a_Tq8F69 41 (6FAM)TGGATACGACAGCCTATC(MGB) miR-29_Tq8F68 42 (6FAM)TGGATACGACAACCGAT(MGB) miR30_Tq8F68 43 (6FAM)CTGGATACGACGCTGC(MGB) miR-34_Tq8F68 44 (6FAM)ATACGACACAACCAGC(MGB) miR-200b_Tq8F67 45 (6FAM)ATACGACCATCATTACC(MGB) miR-323_Tq8F67 46 (6FAM)CTGGATACGACAGAGGT(MGB) miR-324-5Tq8F68 47 (6FAM)ATACGACACACCAATGC(MGB) let7a_Tq8F68 48 (6FAM)TGGATACGACAACTATAC(MGB) Universal  SEQ ID reverse primer ID NO: Reverse primer sequence miR-UP-R67.8 49 GTGCAGGGTCCGAGGT

Example 2

A multiplex (12-plex) assay was performed and the results compared to a corresponding collection of single-plex reactions. Additionally, the effect of the presence or absence of ligase, as well as the presence or absence of reverse transcriptase, was determined. The experiments were performed essentially the same as in Example 1, and the concentration of each linker in the 12-plex reaction was 0.05 uM, thereby resulting in a total linker probe concentration of 0.6 uM. Further, the diluted 12-plex reverse transcription product was split into 12 different PCR amplification reactions, wherein a miRNA forward primer and a universal reverse primer and a detector probe where in each amplification reaction. The miRNA sequences, Forward primers, and TaqMan detector probes are included in Table 2. The results are shown in Table 3.

TABLE 3 Singleplex vs. Multiplex Assay With Or Without T4 DNA Ligase 1-plex Ct 12-plex Ct Ligation + RT 1- vs. 12- miRNA Ligation + RT RT only Ligation + RT RT only vs RT only plex let-7a1 17.8 16.3 17.6 17.0 1.0 −0.3 mir-16 16.0 15.1 16.1 15.3 0.9 −0.1 mir-20 19.3 18.7 19.8 19.5 0.4 −0.6 mir-21 17.0 15.8 17.1 16.3 1.0 −0.3 mir-22 21.6 20.4 21.4 20.7 1.0 −0.1 mir-26a 15.2 14.3 15.6 14.9 0.8 −0.4 mir-29 17.9 16.8 17.7 17.0 0.9 0.0 mir-30a 20.7 19.9 21.2 20.7 0.7 −0.7 mir-34 21.3 20.4 22.0 21.0 0.9 −0.6 mir-200b 19.9 19.2 21.1 20.2 0.8 −1.0 mir-323 32.5 31.2 33.6 32.3 1.3 −1.1 mir-324-5 24.7 23.1 25.0 24.4 1.1 −0.8 Average 20.3 19.3 20.7 19.9 0.9 −0.5

Example 3

An experiment was performed to determine the effect of buffer conditions on reaction performance. In one set of experiments, a commercially available reverse transcription buffer from Applied Biosystems (part number 43400550) was employed in the hybridization and extension reaction. In a corresponding set of experiments, a commercially available T4 DNA ligase buffer (NEB) was employed in the hybridization and extension reaction. The experiments were performed as single-plex format essentially as described for Example 1, and each miRNA was done in triplicate. The results are shown in Table 4, comparing RT buffer (AB part #4340550) vs T4 DNA ligase buffer.

TABLE 4 T4 DNA RT vs RT Buffer Ligase Buffer T4 I II III Mean I II III Mean Buffer let-7a1 22.7 22.8 22.8 22.8 20.8 20.7 20.6 20.7 2.1 mir-16 18.4 18.5 18.6 18.5 17.7 17.8 17.9 17.8 0.7 mir-20 23.6 23.7 23.8 23.7 23.1 23.1 23.0 23.1 0.6 mir-21 20.4 20.4 20.5 20.4 19.4 19.3 19.2 19.3 1.1 mir-22 24.0 23.9 24.1 24.0 22.7 22.7 22.7 22.7 1.3 mir-26a 19.8 19.9 20.1 19.9 18.9 19.0 19.0 18.9 1.0 mir-29 21.3 21.3 21.4 21.3 20.5 20.6 20.5 20.5 0.8 mir-30a 24.4 24.4 24.4 24.4 23.6 23.4 23.6 23.5 0.9 mir-34 24.9 24.8 25.1 25.0 23.0 23.1 23.2 23.1 1.9 mir- 25.8 25.8 25.9 25.9 24.6 24.6 24.8 24.7 1.2 200b mir-323 34.6 34.5 34.8 34.6 34.7 34.2 34.5 34.5 0.2 mir- 26.0 26.0 26.1 26.0 25.4 25.7 25.6 25.6 0.5 324-5 Average 23.8 23.8 24.0 23.9 22.9 22.8 22.9 22.9 1.0

Example 4

An experiment was performed to examine the effect of ligase and kinase in a real-time miRNA amplification reaction. Here, twelve single-plex reactions were performed in duplicate, essentially as described in Example 1. Results are shown in Table 5.

TABLE 5 Ligase & Kinase No Ligase/No Kinase I II Mean I II Mean let-7a1 17.7 17.9 17.8 16.2 16.4 16.3 mir-16 15.9 16.2 16.0 15.0 15.2 15.1 mir-20 19.1 19.6 19.3 18.6 18.9 18.7 mir-21 16.9 17.2 17.0 15.7 15.9 15.8 mir-22 21.4 21.7 21.6 20.3 20.5 20.4 mir-26a 15.0 15.4 15.2 14.3 14.4 14.3 mir-29 17.9 18.0 17.9 16.7 16.8 16.8 mir-30a 20.6 20.8 20.7 19.8 20.0 19.9 mir-34 21.1 21.5 21.3 20.4 20.5 20.4 mir-200b 19.8 20.0 19.9 19.2 19.3 19.2 mir-323 32.3 32.6 32.5 31.1 31.2 31.2 mir-324-5 24.6 24.8 24.7 23.0 23.3 23.1 Average 20.2 20.5 20.3 19.2 19.4 19.3

Example 5

An experiment was performed to determine the effect of sample material on Ct values in a real-time miRNA amplification reaction. Here, cells, GuHCl lysate, Tris lysate, and Purified RNA were compared. The cells were NIH3T3 cells. The Purified RNA was collected using the commercially available mirVana mRNA isolation kit for Ambion (catalog number 1560). A Tris lysate, and a Guanidine lysate (GuHCl) (commercially available from Applied Biosystems), were prepared as follows:

For the Tris lysate, a 1× lysis buffer comprised 10 mM Tris-HCl, pH 8.0, 0.02% Sodium Azide, and 0.03% Tween-20. Trypsinized cells were pelleted by centrifugation at 1500 rpm for 5 minutes. The growth media was removed by aspiration, being careful that the cell pellet was not disturbed. PBS was added to bring the cells to 2×10³ cells/ul. Next 10 ul of cell suspension was mixed with 10 ul of a 2× lysis buffer and spun briefly. The tubes were then immediately incubated for 5 minutes at 95 C, and then immediately placed in a chilled block on ice for 2 minutes. The tubes were then mixed well and spun briefly at full speed before use (or optionally, stored at −20 C).

For the GuHCl lysate, a 1× lysis buffer comprised 2.5M GuHCl, 150 mM MES pH 6.0, 200 mM NaCl, 0.75% Tween-20. Trypsinized cells were pelleted by centrifugation at 1500 rpm for 5 minutes. The growth media was removed by aspiration, being careful that the cell pellet was not disturbed. The cell pellet was then re-suspended in 1×PBS, Ca++ and Mg++ free to bring cells to 2×10⁴ cells/uL. Then, 1 volume of 2× lysis buffer was added. To ensure complete nucleic acid release, this was followed by pipetting up and down ten times, followed by a brief spin. Results are shown in Table 6.

Similar results were obtained for a variety of cell lines, including NIH/3T3, OP9, A549, and HepG2 cells.

TABLE 6 Ct GuHCl Tris Purified miRNA ID Cells lysate lysate RNA let-7a1 24.9 31.3 28.2 31.5 mir-16 22.3 25.2 22.3 24.9 mir-20 22.7 26.0 24.1 26.1 mir-21 21.3 24.2 22.0 24.7 mir-22 30.3 28.6 27.2 28.8 mir-26a 25.6 31.0 27.9 31.4 mir-29 27.2 27.9 26.5 27.4 mir-30a 26.1 32.2 28.9 30.7 mir-34 26.8 30.3 26.4 27.4 mir-200b 40.0 40.0 40.0 40.0 mir-323 30.1 34.7 31.1 31.8 mir-324-5 28.6 29.7 28.3 29.3 Average 27.2 30.1 27.8 29.5

Example 6

An experiment was performed to demonstrate the ability of the reaction to selectively quantity mature miRNA in the presence of precursor miRNA. Here, let-7a miRNA and mir-26b miRNA were queried in both mature form as well as in their precursor form. Experiments were performed essentially as described for Example 1 in the no ligase condition, done in triplicate, with varying amounts of target material as indicated. Results are shown in Table 7. The sequences examined were as follows:

Mature let-7a,  Seq ID NO: 50 UGAGGUAGUAGGUUGUAUAGUU Precursor let-7a,  SEQ ID NO: 51 GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUCUGCCCUGCUAUGGGA UAACUAUACAAUCUACUGUCUUUCCU (Note that the underlined sequences corresponds to the Mature let-7a.) Mature mir-26b, SEQ ID NO: 52 UUCAAGUAAUUCAGGAUAGGU Precursor mir-26b of SEQ ID NO: 53 CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUUGUGUGCUGUCCAGCCU GUUCUCCAUUACUUGGCUCGGGGACCGG (Note that the underlined sequences corresponds to the Mature mir-26b.)

TABLE 7 Mouse lung Synthetic Synthetic RNA miRNA precursor Assay specific for (C_(T)) Target (ng) (fM) (fM) miRNA Precursor Let-7a 0 0 0 40.0 ± 0.0 40.0 ± 0.0 (let-7a3) 0 10 0 24.2 ± 0.3 40.0 ± 0.0 0 100 0 21.0 ± 0.2 40.0 ± 0.0 0 0 10 35.0 ± 1.0 25.0 ± 0.1 0 0 100 31.0 ± 0.1 21.5 ± 0.1 10 0 0 19.1 ± 0.4 40.0 ± 0.0 Mir-26b 0 0 0 40.0 ± 0.0 40.0 ± 0.0 0 10 0 23.1 ± 0.1 40.0 ± 0.0 0 100 0 19.7 ± 0.1 40.0 ± 0.0 0 0 10 32.9 ± 0.4 25.7 ± 0.0 0 0 100 28.9 ± 0.2 22.3 ± 0.0 10 0 0 20.5 ± 0.1 28.0 ± 0.2

Example 7

An experiment was performed on synthetic let-7a miRNA to assess the number of 3′ nucleotides in the 3′ target specific portion of the linker probe that correspond with the 3′ end region of the miRNA. The experiment was performed as essentially as described supra for Example 1 for the no ligase condition, and results are shown in Table 8 as means and standard deviations of Ct values.

TABLE 8 miRNA assay components: let-7a miRNA synthetic target: let-7a No. 3′ ssDNA C_(T) values & statistics linker probe target specific portion bases I II III Average SD 7 29.4 29.1 29.3 29.3 0.1 6 30.1 29.9 30.2 30.1 0.2 5 33.9 33.2 33.8 33.6 0.4 4 40.0 39.2 40.0 39.7 0.4 In some embodiments, 3′ target specific portions of linker probes preferably comprise 5 nucleotides that correspond to the 3′ end region of miRNAs. For example, miR-26a and miR-26b differ by only 2 bases, one of which is the 3′ end nucleotide of miR-26a. Linker probes comprising 5 nucleotides at their 3′ target specific portions can be employed to selectively detect miR-26a versus miR-26b.

Additional strategies for using the linker probes of the present teachings in the context of single step assays, as well as in the context of short primer compositions, can be found in filed U.S. Provisional Application “Compositions, Methods, and Kits for Identifying and Quantitating Small RNA Molecules” by Lao and Straus, as well as in Elfaitouri et al., J. Clin. Virol. 2004, 30(2): 150-156.

The present teachings further contemplate linker probe compositions comprising 3′ target specific portions corresponding to any micro RNA sequence, including but without limitation, those sequences shown in Table 9, including C. elegans (cel), mouse (mmu), human (hsa), Drosophila (dme), rat (mo), and rice (osa).

TABLE 9 SEQ ID NO: cel-let-7 ugagguaguagguuguauaguu 54 cel-lin-4 ucccugagaccucaaguguga 55 cel-miR-1 uggaauguaaagaaguaugua 56 cel-miR-2 uaucacagccagcuuugaugugc 57 cel-miR-34 aggcagugugguuagcugguug 58 cel-miR-35 ucaccggguggaaacuagcagu 59 cel-miR-36 ucaccgggugaaaauucgcaug 60 cel-miR-37 ucaccgggugaacacuugcagu 61 cel-miR-38 ucaccgggagaaaaacuggagu 62 cel-miR-39 ucaccggguguaaaucagcuug 63 cel-miR-40 ucaccggguguacaucagcuaa 64 cel-miR-41 ucaccgggugaaaaaucaccua 65 cel-miR-42 caccggguuaacaucuacag 66 cel-miR-43 uaucacaguuuacuugcugucgc 67 cel-miR-44 ugacuagagacacauucagcu 68 cel-miR-45 ugacuagagacacauucagcu 69 cel-miR-46 ugucauggagucgcucucuuca 70 cel-miR-47 ugucauggaggcgcucucuuca 71 cel-miR-48 ugagguaggcucaguagaugcga 72 cel-miR-49 aagcaccacgagaagcugcaga 73 cel-miR-50 ugauaugucugguauucuuggguu 74 cel-miR-51 uacccguagcuccuauccauguu 75 cel-miR-52 cacccguacauauguuuccgugcu 76 cel-miR-53 cacccguacauuuguuuccgugcu 77 cel-miR-54 uacccguaaucuucauaauccgag 78 cel-miR-55 uacccguauaaguuucugcugag 79 cel-miR-56* uggcggauccauuuuggguugua 80 cel-miR-56 uacccguaauguuuccgcugag 81 cel-miR-57 uacccuguagaucgagcugugugu 82 cel-miR-58 ugagaucguucaguacggcaau 83 cel-miR-59 ucgaaucguuuaucaggaugaug 84 cel-miR-60 uauuaugcacauuuucuaguuca 85 cel-miR-61 ugacuagaaccguuacucaucuc 86 cel-miR-62 ugauauguaaucuagcuuacag 87 cel-miR-63 uaugacacugaagcgaguuggaaa 88 cel-miR-64 uaugacacugaagcguuaccgaa 89 cel-miR-65 uaugacacugaagcguaaccgaa 90 cel-miR-66 caugacacugauuagggauguga 91 cel-miR-67 ucacaaccuccuagaaagaguaga 92 cel-miR-68 ucgaagacucaaaaguguaga 93 cel-miR-69 ucgaaaauuaaaaaguguaga 94 cel-miR-70 uaauacgucguugguguuuccau 95 cel-miR-71 ugaaagacauggguaguga 96 cel-miR-72 aggcaagauguuggcauagc 97 cel-miR-73 uggcaagauguaggcaguucagu 98 cel-miR-74 uggcaagaaauggcagucuaca 99 cel-miR-75 uuaaagcuaccaaccggcuuca 100 cel-miR-76 uucguuguugaugaagccuuga 101 cel-miR-77 uucaucaggccauagcugucca 102 cel-miR-78 uggaggccugguuguuugugc 103 cel-miR-79 auaaagcuagguuaccaaagcu 104 cel-miR-227 agcuuucgacaugauucugaac 105 cel-miR-80 ugagaucauuaguugaaagccga 106 cel-miR-81 ugagaucaucgugaaagcuagu 107 cel-miR-82 ugagaucaucgugaaagccagu 108 cel-miR-83 uagcaccauauaaauucaguaa 109 cel-miR-84 ugagguaguauguaauauugua 110 cel-miR-85 uacaaaguauuugaaaagucgugc 111 cel-miR-86 uaagugaaugcuuugccacaguc 112 cel-miR-87 gugagcaaaguuucaggugu 113 cel-miR-90 ugauauguuguuugaaugcccc 114 cel-miR-124 uaaggcacgcggugaaugcca 115 cel-miR-228 aauggcacugcaugaauucacgg 116 cel-miR-229 aaugacacugguuaucuuuuccaucgu 117 cel-miR-230 guauuaguugugcgaccaggaga 118 cel-miR-231 uaagcucgugaucaacaggcagaa 119 cel-miR-232 uaaaugcaucuuaacugcgguga 120 cel-miR-233 uugagcaaugcgcaugugcggga 121 cel-miR-234 uuauugcucgagaauacccuu 122 cel-miR-235 uauugcacucuccccggccuga 123 cel-miR-236 uaauacugucagguaaugacgcu 124 cel-miR-237 ucccugagaauucucgaacagcuu 125 cel-miR-238 uuuguacuccgaugccauucaga 126 cel-miR-239a uuuguacuacacauagguacugg 127 cel-miR-239b uuguacuacacaaaaguacug 128 cel-miR-240 uacuggcccccaaaucuucgcu 129 cel-miR-241 ugagguaggugcgagaaauga 130 cel-miR-242 uugcguaggccuuugcuucga 131 cel-miR-243 cgguacgaucgcggcgggauauc 132 cel-miR-244 ucuuugguuguacaaagugguaug 133 cel-miR-245 auugguccccuccaaguagcuc 134 cel-miR-246 uuacauguuucggguaggagcu 135 cel-miR-247 ugacuagagccuauucucuucuu 136 cel-miR-248 uacacgugcacggauaacgcuca 137 cel-miR-249 ucacaggacuuuugagcguugc 138 cel-miR-250 ucacagucaacuguuggcaugg 139 cel-miR-251 uuaaguaguggugccgcucuuauu 140 cel-miR-252 uaaguaguagugccgcagguaac 141 cel-miR-253 cacaccucacuaacacugacc 142 cel-miR-254 ugcaaaucuuucgcgacuguagg 143 cel-miR-256 uggaaugcauagaagacugua 144 cel-miR-257 gaguaucaggaguacccaguga 145 cel-miR-258 gguuuugagaggaauccuuuu 146 cel-miR-259 aaaucucauccuaaucuggua 147 cel-miR-260 gugaugucgaacucuuguag 148 cel-miR-261 uagcuuuuuaguuuucacg 149 cel-miR-262 guuucucgauguuuucugau 150 cel-miR-264 ggcgggugguuguuguuaug 151 cel-miR-265 ugagggaggaagggugguau 152 cel-miR-266 aggcaagacuuuggcaaagc 153 cel-miR-267 cccgugaagugucugcugca 154 cel-miR-268 ggcaagaauuagaagcaguuuggu 155 cel-miR-269 ggcaagacucuggcaaaacu 156 cel-miR-270 ggcaugauguagcaguggag 157 cel-miR-271 ucgccgggugggaaagcauu 158 cel-miR-272 uguaggcauggguguuug 159 cel-miR-273 ugcccguacugugucggcug 160 cel-miR-353 caauugccauguguugguauu 161 cel-miR-354 accuuguuuguugcugcuccu 162 cel-miR-355 uuuguuuuagccugagcuaug 163 cel-miR-356 uugagcaacgcgaacaaauca 164 cel-miR-357 uaaaugccagucguugcagga 165 cel-miR-358 caauugguaucccugucaagg 166 cel-miR-359 ucacuggucuuucucugacga 167 cel-miR-360 ugaccguaaucccguucacaa 168 cel-lsy-6 uuuuguaugagacgcauuucg 169 cel-miR-392 uaucaucgaucacgugugauga 170 hsa-let-7a ugagguaguagguuguauaguu 171 hsa-let-7b ugagguaguagguugugugguu 172 hsa-let-7c ugagguaguagguuguaugguu 173 hsa-let-7d agagguaguagguugcauagu 174 hsa-let-7e ugagguaggagguuguauagu 175 hsa-let-7f ugagguaguagauuguauaguu 176 hsa-miR-15a uagcagcacauaaugguuugug 177 hsa-miR-16 uagcagcacguaaauauuggcg 178 hsa-miR-17-5p caaagugcuuacagugcagguagu 179 hsa-miR-17-3p acugcagugaaggcacuugu 180 hsa-miR-18 uaaggugcaucuagugcagaua 181 hsa-miR-19a ugugcaaaucuaugcaaaacuga 182 hsa-miR-19b ugugcaaauccaugcaaaacuga 183 hsa-miR-20 uaaagugcuuauagugcaggua 184 hsa-miR-21 uagcuuaucagacugauguuga 185 hsa-miR-22 aagcugccaguugaagaacugu 186 hsa-miR-23a aucacauugccagggauuucc 187 hsa-miR-189 gugccuacugagcugauaucagu 188 hsa-miR-24 uggcucaguucagcaggaacag 189 hsa-miR-25 cauugcacuugucucggucuga 190 hsa-miR-26a uucaaguaauccaggauaggcu 191 hsa-miR-26b uucaaguaauucaggauaggu 192 hsa-miR-27a uucacaguggcuaaguuccgcc 193 hsa-miR-28 aaggagcucacagucuauugag 194 hsa-miR-29a cuagcaccaucugaaaucgguu 195 hsa-miR-30a* uguaaacauccucgacuggaagc 196 hsa-miR-30a cuuucagucggauguuugcagc 197 hsa-miR-31 ggcaagaugcuggcauagcug 198 hsa-miR-32 uauugcacauuacuaaguugc 199 hsa-miR-33 gugcauuguaguugcauug 200 hsa-miR-92 uauugcacuugucccggccugu 201 hsa-miR-93 aaagugcuguucgugcagguag 202 hsa-miR-95 uucaacggguauuuauugagca 203 hsa-miR-96 uuuggcacuagcacauuuuugc 204 hsa-miR-98 ugagguaguaaguuguauuguu 205 hsa-miR-99a aacccguagauccgaucuugug 206 hsa-miR-100 aacccguagauccgaacuugug 207 hsa-miR-101 uacaguacugugauaacugaag 208 hsa-miR-29b uagcaccauuugaaaucagu 209 hsa-miR-103 agcagcauuguacagggcuauga 210 hsa-miR-105 ucaaaugcucagacuccugu 211 hsa-miR-106a aaaagugcuuacagugcagguagc 212 hsa-miR-107 agcagcauuguacagggcuauca 213 hsa-miR-192 cugaccuaugaauugacagcc 214 hsa-miR-196 uagguaguuucauguuguugg 215 hsa-miR-197 uucaccaccuucuccacccagc 216 hsa-miR-198 gguccagaggggagauagg 217 hsa-miR-199a cccaguguucagacuaccuguuc 218 hsa-miR-199a* uacaguagucugcacauugguu 219 hsa-miR-208 auaagacgagcaaaaagcuugu 220 hsa-miR-148a ucagugcacuacagaacuuugu 221 hsa-miR-30c uguaaacauccuacacucucagc 222 hsa-miR-30d uguaaacauccccgacuggaag 223 hsa-miR-139 ucuacagugcacgugucu 224 hsa-miR-147 guguguggaaaugcuucugc 225 hsa-miR-7 uggaagacuagugauuuuguu 226 hsa-miR-10a uacccuguagauccgaauuugug 227 hsa-miR-10b uacccuguagaaccgaauuugu 228 hsa-miR-34a uggcagugucuuagcugguugu 229 hsa-miR-181a aacauucaacgcugucggugagu 230 hsa-miR-181b aacauucauugcugucgguggguu 231 hsa-miR-181c aacauucaaccugucggugagu 232 hsa-miR-182 uuuggcaaugguagaacucaca 233 hsa-miR-182* ugguucuagacuugccaacua 234 hsa-miR-183 uauggcacugguagaauucacug 235 hsa-miR-187 ucgugucuuguguugcagccg 236 hsa-miR-199b cccaguguuuagacuaucuguuc 237 hsa-miR-203 gugaaauguuuaggaccacuag 238 hsa-miR-204 uucccuuugucauccuaugccu 239 hsa-miR-205 uccuucauuccaccggagucug 240 hsa-miR-210 cugugcgugugacagcggcug 241 hsa-miR-211 uucccuuugucauccuucgccu 242 hsa-miR-212 uaacagucuccagucacggcc 243 hsa-miR-213 accaucgaccguugauuguacc 244 hsa-miR-214 acagcaggcacagacaggcag 245 hsa-miR-215 augaccuaugaauugacagac 246 hsa-miR-216 uaaucucagcuggcaacugug 247 hsa-miR-217 uacugcaucaggaacugauuggau 248 hsa-miR-218 uugugcuugaucuaaccaugu 249 hsa-miR-219 ugauuguccaaacgcaauucu 250 hsa-miR-220 ccacaccguaucugacacuuu 251 hsa-miR-221 agcuacauugucugcuggguuuc 252 hsa-miR-222 agcuacaucuggcuacugggucuc 253 hsa-miR-223 ugucaguuugucaaauacccc 254 hsa-miR-224 caagucacuagugguuccguuua 255 hsa-miR-200b cucuaauacugccugguaaugaug 256 hsa-let-7g ugagguaguaguuuguacagu 257 hsa-let-7i ugagguaguaguuugugcu 258 hsa-miR-1 uggaauguaaagaaguaugua 259 hsa-miR-15b uagcagcacaucaugguuuaca 260 hsa-miR-23b aucacauugccagggauuaccac 261 hsa-miR-27b uucacaguggcuaaguucug 262 hsa-miR-30b uguaaacauccuacacucagc 263 hsa-miR-122a uggagugugacaaugguguuugu 264 hsa-miR-124a uuaaggcacgcggugaaugcca 265 hsa-miR-125b ucccugagacccuaacuuguga 266 hsa-miR-128a ucacagugaaccggucucuuuu 267 hsa-miR-130a cagugcaauguuaaaagggc 268 hsa-miR-132 uaacagucuacagccauggucg 269 hsa-miR-133a uugguccccuucaaccagcugu 270 hsa-miR-135a uauggcuuuuuauuccuauguga 271 hsa-miR-137 uauugcuuaagaauacgcguag 272 hsa-miR-138 agcugguguugugaauc 273 hsa-miR-140 agugguuuuacccuaugguag 274 hsa-miR-141 aacacugucugguaaagaugg 275 hsa-miR-142-5p cauaaaguagaaagcacuac 276 hsa-miR-142-3p uguaguguuuccuacuuuaugga 277 hsa-miR-143 ugagaugaagcacuguagcuca 278 hsa-miR-144 uacaguauagaugauguacuag 279 hsa-miR-145 guccaguuuucccaggaaucccuu 280 hsa-miR-152 ucagugcaugacagaacuugg 281 hsa-miR-153 uugcauagucacaaaaguga 282 hsa-miR-191 caacggaaucccaaaagcagcu 283 hsa-miR-9 ucuuugguuaucuagcuguauga 284 hsa-miR-9* uaaagcuagauaaccgaaagu 285 hsa-miR-125a ucccugagacccuuuaaccugug 286 hsa-miR-126* cauuauuacuuuugguacgcg 287 hsa-miR-126 ucguaccgugaguaauaaugc 288 hsa-miR-127 ucggauccgucugagcuuggcu 289 hsa-miR-129 cuuuuugcggucugggcuugc 290 hsa-miR-134 ugugacugguugaccagaggg 291 hsa-miR-136 acuccauuuguuuugaugaugga 292 hsa-miR-146 ugagaacugaauuccauggguu 293 hsa-miR-149 ucuggcuccgugucuucacucc 294 hsa-miR-150 ucucccaacccuuguaccagug 295 hsa-miR-154 uagguuauccguguugccuucg 296 hsa-miR-184 uggacggagaacugauaagggu 297 hsa-miR-185 uggagagaaaggcaguuc 298 hsa-miR-186 caaagaauucuccuuuugggcuu 299 hsa-miR-188 caucccuugcaugguggagggu 300 hsa-miR-190 ugauauguuugauauauuaggu 301 hsa-miR-193 aacuggccuacaaagucccag 302 hsa-miR-194 uguaacagcaacuccaugugga 303 hsa-miR-195 uagcagcacagaaauauuggc 304 hsa-miR-206 uggaauguaaggaagugugugg 305 hsa-miR-320 aaaagcuggguugagagggcgaa 306 hsa-miR-321 uaagccagggauuguggguuc 307 hsa-miR-200c aauacugccggguaaugaugga 308 hsa-miR-155 uuaaugcuaaucgugauagggg 309 hsa-miR-128b ucacagugaaccggucucuuuc 310 hsa-miR-106b uaaagugcugacagugcagau 311 hsa-miR-29c uagcaccauuugaaaucgguua 312 hsa-miR-200a uaacacugucugguaacgaugu 313 hsa-miR-302 uaagugcuuccauguuuugguga 314 hsa-miR-34b aggcagugucauuagcugauug 315 hsa-miR-34c aggcaguguaguuagcugauug 316 hsa-miR-299 ugguuuaccgucccacauacau 317 hsa-miR-301 cagugcaauaguauugucaaagc 318 hsa-miR-99b cacccguagaaccgaccuugcg 319 hsa-miR-296 agggcccccccucaauccugu 320 hsa-miR-130b cagugcaaugaugaaagggcau 321 hsa-miR-30e uguaaacauccuugacugga 322 hsa-miR-340 uccgucucaguuacuuuauagcc 323 hsa-miR-330 gcaaagcacacggccugcagaga 324 hsa-miR-328 cuggcccucucugcccuuccgu 325 hsa-miR-342 ucucacacagaaaucgcacccguc 326 hsa-miR-337 uccagcuccuauaugaugccuuu 327 hsa-miR-323 gcacauuacacggucgaccucu 328 hsa-miR-326 ccucugggcccuuccuccag 329 hsa-miR-151 acuagacugaagcuccuugagg 330 hsa-miR-135b uauggcuuuucauuccuaugug 331 hsa-miR-148b ucagugcaucacagaacuuugu 332 hsa-miR-331 gccccugggccuauccuagaa 333 hsa-miR-324-5p cgcauccccuagggcauuggugu 334 hsa-miR-324-3p ccacugccccaggugcugcugg 335 hsa-miR-338 uccagcaucagugauuuuguuga 336 hsa-miR-339 ucccuguccuccaggagcuca 337 hsa-miR-335 ucaagagcaauaacgaaaaaugu 338 hsa-miR-133b uugguccccuucaaccagcua 339 osa-miR156 ugacagaagagagugagcac 340 osa-miR160 ugccuggcucccuguaugcca 341 osa-miR162 ucgauaaaccucugcauccag 342 osa-miR164 uggagaagcagggcacgugca 343 osa-miR166 ucggaccaggcuucauucccc 344 osa-miR167 ugaagcugccagcaugaucua 345 osa-miR169 cagccaaggaugacuugccga 346 osa-miR171 ugauugagccgcgccaauauc 347 mmu-let-7g ugagguaguaguuuguacagu 348 mmu-let-7i ugagguaguaguuugugcu 349 mmu-miR-1 uggaauguaaagaaguaugua 350 mmu-miR-15b uagcagcacaucaugguuuaca 351 mmu-miR-23b aucacauugccagggauuaccac 352 mmu-miR-27b uucacaguggcuaaguucug 353 mmu-miR-29b uagcaccauuugaaaucagugu 354 mmu-miR-30a* uguaaacauccucgacuggaagc 355 mmu-miR-30a cuuucagucggauguuugcagc 356 mmu-miR-30b uguaaacauccuacacucagc 357 mmu-miR-99a acccguagauccgaucuugu 358 mmu-miR-99b cacccguagaaccgaccuugcg 359 mmu-miR-101 uacaguacugugauaacuga 360 mmu-miR-124a uuaaggcacgcggugaaugcca 361 mmu-miR-125a ucccugagacccuuuaaccugug 362 mmu-miR-125b ucccugagacccuaacuuguga 363 mmu-miR-126* cauuauuacuuuugguacgcg 364 mmu-miR-126 ucguaccgugaguaauaaugc 365 mmu-miR-127 ucggauccgucugagcuuggcu 366 mmu-miR-128a ucacagugaaccggucucuuuu 367 mmu-miR-130a cagugcaauguuaaaagggc 368 mmu-miR-9 ucuuugguuaucuagcuguauga 369 mmu-miR-9* uaaagcuagauaaccgaaagu 370 mmu-miR-132 uaacagucuacagccauggucg 371 mmu-miR-133a uugguccccuucaaccagcugu 372 mmu-miR-134 ugugacugguugaccagaggg 373 mmu-miR-135a uauggcuuuuuauuccuauguga 374 mmu-miR-136 acuccauuuguuuugaugaugga 375 mmu-miR-137 uauugcuuaagaauacgcguag 376 mmu-miR-138 agcugguguugugaauc 377 mmu-miR-140 agugguuuuacccuaugguag 378 mmu-miR-141 aacacugucugguaaagaugg 379 mmu-miR-142-5p cauaaaguagaaagcacuac 380 mmu-miR-142-3p uguaguguuuccuacuuuaugg 381 mmu-miR-144 uacaguauagaugauguacuag 382 mmu-miR-145 guccaguuuucccaggaaucccuu 383 mmu-miR-146 ugagaacugaauuccauggguu 384 mmu-miR-149 ucuggcuccgugucuucacucc 385 mmu-miR-150 ucucccaacccuuguaccagug 386 mmu-miR-151 cuagacugaggcuccuugagg 387 mmu-miR-152 ucagugcaugacagaacuugg 388 mmu-miR-153 uugcauagucacaaaaguga 389 mmu-miR-154 uagguuauccguguugccuucg 390 mmu-miR-155 uuaaugcuaauugugauagggg 391 mmu-miR-10b cccuguagaaccgaauuugugu 392 mmu-miR-129 cuuuuugcggucugggcuugcu 393 mmu-miR-181a aacauucaacgcugucggugagu 394 mmu-miR-182 uuuggcaaugguagaacucaca 395 mmu-miR-183 uauggcacugguagaauucacug 396 mmu-miR-184 uggacggagaacugauaagggu 397 mmu-miR-185 uggagagaaaggcaguuc 398 mmu-miR-186 caaagaauucuccuuuugggcuu 399 mmu-miR-187 ucgugucuuguguugcagccgg 400 mmu-miR-188 caucccuugcaugguggagggu 401 mmu-miR-189 gugccuacugagcugauaucagu 402 mmu-miR-24 uggcucaguucagcaggaacag 403 mmu-miR-190 ugauauguuugauauauuaggu 404 mmu-miR-191 caacggaaucccaaaagcagcu 405 mmu-miR-193 aacuggccuacaaagucccag 406 mmu-miR-194 uguaacagcaacuccaugugga 407 mmu-miR-195 uagcagcacagaaauauuggc 408 mmu-miR-199a cccaguguucagacuaccuguuc 409 mmu-miR-199a* uacaguagucugcacauugguu 410 mmu-miR-200b uaauacugccugguaaugaugac 411 mmu-miR-201 uacucaguaaggcauuguucu 412 mmu-miR-202 agagguauagcgcaugggaaga 413 mmu-miR-203 ugaaauguuuaggaccacuag 414 mmu-miR-204 uucccuuugucauccuaugccug 415 mmu-miR-205 uccuucauuccaccggagucug 416 mmu-miR-206 uggaauguaaggaagugugugg 417 mmu-miR-207 gcuucuccuggcucuccucccuc 418 mmu-miR-122a uggagugugacaaugguguuugu 419 mmu-miR-143 ugagaugaagcacuguagcuca 420 mmu-miR-30e uguaaacauccuugacugga 421 mmu-miR-290 cucaaacuaugggggcacuuuuu 422 mmu-miR-291-5p caucaaaguggaggcccucucu 423 mmu-miR-291-3p aaagugcuuccacuuugugugcc 424 mmu-miR-292-5p acucaaacugggggcucuuuug 425 mmu-miR-292-3p aagugccgccagguuuugagugu 426 mmu-miR-293 agugccgcagaguuuguagugu 427 mmu-miR-294 aaagugcuucccuuuugugugu 428 mmu-miR-295 aaagugcuacuacuuuugagucu 429 mmu-miR-296 agggcccccccucaauccugu 430 mmu-miR-297 auguaugugugcaugugcaug 431 mmu-miR-298 ggcagaggagggcuguucuucc 432 mmu-miR-299 ugguuuaccgucccacauacau 433 mmu-miR-300 uaugcaagggcaagcucucuuc 434 mmu-miR-301 cagugcaauaguauugucaaagc 435 mmu-miR-302 uaagugcuuccauguuuugguga 436 mmu-miR-34c aggcaguguaguuagcugauugc 437 mmu-miR-34b uaggcaguguaauuagcugauug 438 mmu-let-7d agagguaguagguugcauagu 439 mmu-let-7d* cuauacgaccugcugccuuucu 440 mmu-miR-106a caaagugcuaacagugcaggua 441 mmu-miR-106b uaaagugcugacagugcagau 442 mmu-miR-130b cagugcaaugaugaaagggcau 443 mmu-miR-19b ugugcaaauccaugcaaaacuga 444 mmu-miR-30c uguaaacauccuacacucucagc 445 mmu-miR-30d uguaaacauccccgacuggaag 446 mmu-miR-148a ucagugcacuacagaacuuugu 447 mmu-miR-192 cugaccuaugaauugaca 448 mmu-miR-196 uagguaguuucauguuguugg 449 mmu-miR-200a uaacacugucugguaacgaugu 450 mmu-miR-208 auaagacgagcaaaaagcuugu 451 mmu-let-7a ugagguaguagguuguauaguu 452 mmu-let-7b ugagguaguagguugugugguu 453 mmu-let-7c ugagguaguagguuguaugguu 454 mmu-let-7e ugagguaggagguuguauagu 455 mmu-let-7f ugagguaguagauuguauaguu 456 mmu-miR-15a uagcagcacauaaugguuugug 457 mmu-miR-16 uagcagcacguaaauauuggcg 458 mmu-miR-18 uaaggugcaucuagugcagaua 459 mmu-miR-20 uaaagugcuuauagugcagguag 460 mmu-miR-21 uagcuuaucagacugauguuga 461 mmu-miR-22 aagcugccaguugaagaacugu 462 mmu-miR-23a aucacauugccagggauuucc 463 mmu-miR-26a uucaaguaauccaggauaggcu 464 mmu-miR-26b uucaaguaauucaggauagguu 465 mmu-miR-29a cuagcaccaucugaaaucgguu 466 mmu-miR-29c uagcaccauuugaaaucgguua 467 mmu-miR-27a uucacaguggcuaaguuccgc 468 mmu-miR-31 aggcaagaugcuggcauagcug 469 mmu-miR-92 uauugcacuugucccggccug 470 mmu-miR-93 caaagugcuguucgugcagguag 471 mmu-miR-96 uuuggcacuagcacauuuuugcu 472 mmu-miR-34a uggcagugucuuagcugguuguu 473 mmu-miR-98 ugagguaguaaguuguauuguu 474 mmu-miR-103 agcagcauuguacagggcuauga 475 mmu-miR-323 gcacauuacacggucgaccucu 476 mmu-miR-324-5p cgcauccccuagggcauuggugu 477 mmu-miR-324-3p ccacugccccaggugcugcugg 478 mmu-miR-325 ccuaguaggugcucaguaagugu 479 mmu-miR-326 ccucugggcccuuccuccagu 480 mmu-miR-328 cuggcccucucugcccuuccgu 481 mmu-miR-329 aacacacccagcuaaccuuuuu 482 mmu-miR-330 gcaaagcacagggccugcagaga 483 mmu-miR-331 gccccugggccuauccuagaa 484 mmu-miR-337 uucagcuccuauaugaugccuuu 485 mmu-miR-338 uccagcaucagugauuuuguuga 486 mmu-miR-339 ucccuguccuccaggagcuca 487 mmu-miR-340 uccgucucaguuacuuuauagcc 488 mmu-miR-341 ucgaucggucggucggucagu 489 mmu-miR-342 ucucacacagaaaucgcacccguc 490 mmu-miR-344 ugaucuagccaaagccugacugu 491 mmu-miR-345 ugcugaccccuaguccagugc 492 mmu-miR-346 ugucugcccgagugccugccucu 493 mmu-miR-350 uucacaaagcccauacacuuucac 494 mmu-miR-135b uauggcuuuucauuccuaugug 495 mmu-miR-101b uacaguacugugauagcugaag 496 mmu-miR-107 agcagcauuguacagggcuauca 497 mmu-miR-10a uacccuguagauccgaauuugug 498 mmu-miR-17-5p caaagugcuuacagugcagguagu 499 mmu-miR-17-3p acugcagugagggcacuugu 500 mmu-miR-19a ugugcaaaucuaugcaaaacuga 501 mmu-miR-25 cauugcacuugucucggucuga 502 mmu-miR-28 aaggagcucacagucuauugag 503 mmu-miR-32 uauugcacauuacuaaguugc 504 mmu-miR-100 aacccguagauccgaacuugug 505 mmu-miR-139 ucuacagugcacgugucu 506 mmu-miR-200c aauacugccggguaaugaugga 507 mmu-miR-210 cugugcgugugacagcggcug 508 mmu-miR-212 uaacagucuccagucacggcc 509 mmu-miR-213 accaucgaccguugauuguacc 510 mmu-miR-214 acagcaggcacagacaggcag 511 mmu-miR-216 uaaucucagcuggcaacugug 512 mmu-miR-218 uugugcuugaucuaaccaugu 513 mmu-miR-219 ugauuguccaaacgcaauucu 514 mmu-miR-223 ugucaguuugucaaauacccc 515 mmu-miR-320 aaaagcuggguugagagggcgaa 516 mmu-miR-321 uaagccagggauuguggguuc 517 mmu-miR-33 gugcauuguaguugcauug 518 mmu-miR-211 uucccuuugucauccuuugccu 519 mmu-miR-221 agcuacauugucugcuggguuu 520 mmu-miR-222 agcuacaucuggcuacugggucu 521 mmu-miR-224 uaagucacuagugguuccguuua 522 mmu-miR-199b cccaguguuuagacuaccuguuc 523 mmu-miR-181b aacauucauugcugucgguggguu 524 mmu-miR-181c aacauucaaccugucggugagu 525 mmu-miR-128b ucacagugaaccggucucuuuc 526 mmu-miR-7 uggaagacuagugauuuuguu 527 mmu-miR-7b uggaagacuugugauuuuguu 528 mmu-miR-217 uacugcaucaggaacugacuggau 529 mmu-miR-133b uugguccccuucaaccagcua 530 mmu-miR-215 augaccuaugauuugacagac 531 dme-miR-1 uggaauguaaagaaguauggag 532 dme-miR-2a uaucacagccagcuuugaugagc 533 dme-miR-2b uaucacagccagcuuugaggagc 534 dme-miR-3 ucacugggcaaagugugucuca 535 dme-miR-4 auaaagcuagacaaccauuga 536 dme-miR-5 aaaggaacgaucguugugauaug 537 dme-miR-6 uaucacaguggcuguucuuuuu 538 dme-miR-7 uggaagacuagugauuuuguugu 539 dme-miR-8 uaauacugucagguaaagauguc 540 dme-miR-9a ucuuugguuaucuagcuguauga 541 dme-miR-10 acccuguagauccgaauuugu 542 dme-miR-11 caucacagucugaguucuugc 543 dme-miR-12 ugaguauuacaucagguacuggu 544 dme-miR-13a uaucacagccauuuugaugagu 545 dme-miR-13b uaucacagccauuuugacgagu 546 dme-miR-14 ucagucuuuuucucucuccua 547 dme-miR-263a guuaauggcacuggaagaauucac 548 dme-miR-184* ccuuaucauucucucgccccg 549 dme-miR-184 uggacggagaacugauaagggc 550 dme-miR-274 uuuugugaccgacacuaacggguaau 551 dme-miR-275 ucagguaccugaaguagcgcgcg 552 dme-miR-92a cauugcacuugucccggccuau 553 dme-miR-219 ugauuguccaaacgcaauucuug 554 dme-miR-276a* cagcgagguauagaguuccuacg 555 dme-miR-276a uaggaacuucauaccgugcucu 556 dme-miR-277 uaaaugcacuaucugguacgaca 557 dme-miR-278 ucggugggacuuucguccguuu 558 dme-miR-133 uugguccccuucaaccagcugu 559 dme-miR-279 ugacuagauccacacucauuaa 560 dme-miR-33 aggugcauuguagucgcauug 561 dme-miR-280 uguauuuacguugcauaugaaaugaua 562 dme-miR-281-1* aagagagcuguccgucgacagu 563 dme-miR-281 ugucauggaauugcucucuuugu 564 dme-miR-282 aaucuagccucuacuaggcuuugucugu 565 dme-miR-283 uaaauaucagcugguaauucu 566 dme-miR-284 ugaagucagcaacuugauuccagcaau 567 ug dme-miR-281-2* aagagagcuauccgucgacagu 568 dme-miR-34 uggcagugugguuagcugguug 569 dme-miR-124 uaaggcacgcggugaaugccaag 570 dme-miR-79 uaaagcuagauuaccaaagcau 571 dme-miR-276b* cagcgagguauagaguuccuacg 572 dme-miR-276b uaggaacuuaauaccgugcucu 573 dme-miR-210 uugugcgugugacagcggcua 574 dme-miR-285 uagcaccauucgaaaucagugc 575 dme-miR-100 aacccguaaauccgaacuugug 576 dme-miR-92b aauugcacuagucccggccugc 577 dme-miR-286 ugacuagaccgaacacucgugcu 578 dme-miR-287 uguguugaaaaucguuugcac 579 dme-miR-87 uugagcaaaauuucaggugug 580 dme-miR-263b cuuggcacugggagaauucac 581 dme-miR-288 uuucaugucgauuucauuucaug 582 dme-miR-289 uaaauauuuaaguggagccugcgacu 583 dme-bantam ugagaucauuuugaaagcugauu 584 dme-miR-303 uuuagguuucacaggaaacuggu 585 dme-miR-31b uggcaagaugucggaauagcug 586 dme-miR-304 uaaucucaauuuguaaaugugag 587 dme-miR-305 auuguacuucaucaggugcucug 588 dme-miR-9c ucuuugguauucuagcuguaga 589 dme-miR-306 ucagguacuuagugacucucaa 590 dme-miR-306* gggggucacucugugccugugc 591 dme-miR-9b ucuuuggugauuuuagcuguaug 592 dme-let-7 ugagguaguagguuguauagu 593 dme-miR-125 ucccugagacccuaacuuguga 594 dme-miR-307 ucacaaccuccuugagugag 595 dme-miR-308 aaucacaggauuauacugugag 596 dme-miR-31a uggcaagaugucggcauagcuga 597 dme-miR-309 gcacuggguaaaguuuguccua 598 dme-miR-310 uauugcacacuucccggccuuu 599 dme-miR-311 uauugcacauucaccggccuga 600 dme-miR-312 uauugcacuugagacggccuga 601 dme-miR-313 uauugcacuuuucacagcccga 602 dme-miR-314 uauucgagccaauaaguucgg 603 dme-miR-315 uuuugauuguugcucagaaagc 604 dme-miR-316 ugucuuuuuccgcuuacuggcg 605 dme-miR-317 ugaacacagcuggugguauccagu 606 dme-miR-318 ucacugggcuuuguuuaucuca 607 dme-miR-2c uaucacagccagcuuugaugggc 608 dme-miR-iab-4-5p acguauacugaauguauccuga 609 dme-miR-iab-4-3p cgguauaccuucaguauacguaac 610 rno-miR-322 aaacaugaagcgcugcaaca 611 rno-miR-323 gcacauuacacggucgaccucu 612 rno-miR-301 cagugcaauaguauugucaaagcau 613 rno-miR-324-5p cgcauccccuagggcauuggugu 614 rno-miR-324-3p ccacugccccaggugcugcugg 615 rno-miR-325 ccuaguaggugcucaguaagugu 616 rno-miR-326 ccucugggcccuuccuccagu 617 rno-let-7d agagguaguagguugcauagu 618 rno-let-7d* cuauacgaccugcugccuuucu 619 rno-miR-328 cuggcccucucugcccuuccgu 620 rno-miR-329 aacacacccagcuaaccuuuuu 621 rno-miR-330 gcaaagcacagggccugcagaga 622 rno-miR-331 gccccugggccuauccuagaa 623 rno-miR-333 guggugugcuaguuacuuuu 624 rno-miR-140 agugguuuuacccuaugguag 625 rno-miR-140* uaccacaggguagaaccacggaca 626 rno-miR-336 ucacccuuccauaucuagucu 627 rno-miR-337 uucagcuccuauaugaugccuuu 628 rno-miR-148b ucagugcaucacagaacuuugu 629 rno-miR-338 uccagcaucagugauuuuguuga 630 rno-miR-339 ucccuguccuccaggagcuca 631 rno-miR-341 ucgaucggucggucggucagu 632 rno-miR-342 ucucacacagaaaucgcacccguc 633 rno-miR-344 ugaucuagccaaagccugaccgu 634 rno-miR-345 ugcugaccccuaguccagugc 635 rno-miR-346 ugucugccugagugccugccucu 636 rno-miR-349 cagcccugcugucuuaaccucu 637 rno-miR-129 cuuuuugcggucugggcuugcu 638 rno-miR-129* aagcccuuaccccaaaaagcau 639 rno-miR-20 uaaagugcuuauagugcagguag 640 rno-miR-20* acugcauuacgagcacuuaca 641 rno-miR-350 uucacaaagcccauacacuuucac 642 rno-miR-7 uggaagacuagugauuuuguu 643 rno-miR-7* caacaaaucacagucugccaua 644 rno-miR-351 ucccugaggagcccuuugagccug 645 rno-miR-135b uauggcuuuucauuccuaugug 646 rno-miR-151* ucgaggagcucacagucuagua 647 rno-miR-151 acuagacugaggcuccuugagg 648 rno-miR-101b uacaguacugugauagcugaag 649 rno-let-7a ugagguaguagguuguauaguu 650 rno-let-7b ugagguaguagguugugugguu 651 rno-let-7c ugagguaguagguuguaugguu 652 rno-let-7e ugagguaggagguuguauagu 653 rno-let-7f ugagguaguagauuguauaguu 654 rno-let-7i ugagguaguaguuugugcu 655 rno-miR-7b uggaagacuugugauuuuguu 656 rno-miR-9 ucuuugguuaucuagcuguauga 657 rno-miR-10a uacccuguagauccgaauuugug 658 rno-miR-10b uacccuguagaaccgaauuugu 659 rno-miR-15b uagcagcacaucaugguuuaca 660 rno-miR-16 uagcagcacguaaauauuggcg 661 rno-miR-17 caaagugcuuacagugcagguagu 662 rno-miR-18 uaaggugcaucuagugcagaua 663 rno-miR-19b ugugcaaauccaugcaaaacuga 664 rno-miR-19a ugugcaaaucuaugcaaaacuga 665 rno-miR-21 uagcuuaucagacugauguuga 666 rno-miR-22 aagcugccaguugaagaacugu 667 rno-miR-23a aucacauugccagggauuucc 668 rno-miR-23b aucacauugccagggauuaccac 669 rno-miR-24 uggcucaguucagcaggaacag 670 rno-miR-25 cauugcacuugucucggucuga 671 rno-miR-26a uucaaguaauccaggauaggcu 672 rno-miR-26b uucaaguaauucaggauagguu 673 rno-miR-27b uucacaguggcuaaguucug 674 rno-miR-27a uucacaguggcuaaguuccgc 675 rno-miR-28 aaggagcucacagucuauugag 676 rno-miR-29b uagcaccauuugaaaucagugu 677 rno-miR-29a cuagcaccaucugaaaucgguu 678 rno-miR-29c uagcaccauuugaaaucgguua 679 rno-miR-30c uguaaacauccuacacucucagc 680 rno-miR-30e uguaaacauccuugacugga 681 rno-miR-30b uguaaacauccuacacucagc 682 rno-miR-30d uguaaacauccccgacuggaag 683 rno-miR-30a cuuucagucggauguuugcagc 684 rno-miR-31 aggcaagaugcuggcauagcug 685 rno-miR-32 uauugcacauuacuaaguugc 686 rno-miR-33 gugcauuguaguugcauug 687 rno-miR-34b uaggcaguguaauuagcugauug 688 rno-miR-34c aggcaguguaguuagcugauugc 689 rno-miR-34a uggcagugucuuagcugguuguu 690 rno-miR-92 uauugcacuugucccggccug 691 rno-miR-93 caaagugcuguucgugcagguag 692 rno-miR-96 uuuggcacuagcacauuuuugcu 693 rno-miR-98 ugagguaguaaguuguauuguu 694 rno-miR-99a aacccguagauccgaucuugug 695 rno-miR-99b cacccguagaaccgaccuugcg 696 rno-miR-100 aacccguagauccgaacuugug 697 rno-miR-101 uacaguacugugauaacugaag 698 rno-miR-103 agcagcauuguacagggcuauga 699 rno-miR-106b uaaagugcugacagugcagau 700 rno-miR-107 agcagcauuguacagggcuauca 701 rno-miR-122a uggagugugacaaugguguuugu 702 rno-miR-124a uuaaggcacgcggugaaugcca 703 rno-miR-125a ucccugagacccuuuaaccugug 704 rno-miR-125b ucccugagacccuaacuuguga 705 rno-miR-126* cauuauuacuuuugguacgcg 706 rno-miR-126 ucguaccgugaguaauaaugc 707 rno-miR-127 ucggauccgucugagcuuggcu 708 rno-miR-128a ucacagugaaccggucucuuuu 709 rno-miR-128b ucacagugaaccggucucuuuc 710 rno-miR-130a cagugcaauguuaaaagggc 711 rno-miR-130b cagugcaaugaugaaagggcau 712 rno-miR-132 uaacagucuacagccauggucg 713 rno-miR-133a uugguccccuucaaccagcugu 714 rno-miR-134 ugugacugguugaccagaggg 715 rno-miR-135a uauggcuuuuuauuccuauguga 716 rno-miR-136 acuccauuuguuuugaugaugga 717 rno-miR-137 uauugcuuaagaauacgcguag 718 rno-miR-138 agcugguguugugaauc 719 rno-miR-139 ucuacagugcacgugucu 720 rno-miR-141 aacacugucugguaaagaugg 721 rno-miR-142-5p cauaaaguagaaagcacuac 722 rno-miR-142-3p uguaguguuuccuacuuuaugga 723 rno-miR-143 ugagaugaagcacuguagcuca 724 rno-miR-144 uacaguauagaugauguacuag 725 rno-miR-145 guccaguuuucccaggaaucccuu 726 rno-miR-146 ugagaacugaauuccauggguu 727 rno-miR-150 ucucccaacccuuguaccagug 728 rno-miR-152 ucagugcaugacagaacuugg 729 rno-miR-153 uugcauagucacaaaaguga 730 rno-miR-154 uagguuauccguguugccuucg 731 rno-miR-181c aacauucaaccugucggugagu 732 rno-miR-181a aacauucaacgcugucggugagu 733 rno-miR-181b aacauucauugcugucgguggguu 734 rno-miR-183 uauggcacugguagaauucacug 735 rno-miR-184 uggacggagaacugauaagggu 736 rno-miR-185 uggagagaaaggcaguuc 737 rno-miR-186 caaagaauucuccuuuugggcuu 738 rno-miR-187 ucgugucuuguguugcagccg 739 rno-miR-190 ugauauguuugauauauuaggu 740 rno-miR-191 caacggaaucccaaaagcagcu 741 rno-miR-192 cugaccuaugaauugacagcc 742 rno-miR-193 aacuggccuacaaagucccag 743 rno-miR-194 uguaacagcaacuccaugugga 744 rno-miR-195 uagcagcacagaaauauuggc 745 rno-miR-196 uagguaguuucauguuguugg 746 rno-miR-199a cccaguguucagacuaccuguuc 747 rno-miR-200c aauacugccggguaaugaugga 748 rno-miR-200a uaacacugucugguaacgaugu 749 rno-miR-200b cucuaauacugccugguaaugaug 750 rno-miR-203 gugaaauguuuaggaccacuag 751 rno-miR-204 uucccuuugucauccuaugccu 752 rno-miR-205 uccuucauuccaccggagucug 753 rno-miR-206 uggaauguaaggaagugugugg 754 rno-miR-208 auaagacgagcaaaaagcuugu 755 rno-miR-210 cugugcgugugacagcggcug 756 rno-miR-211 uucccuuugucauccuuugccu 757 rno-miR-212 uaacagucuccagucacggcc 758 rno-miR-213 accaucgaccguugauuguacc 759 rno-miR-214 acagcaggcacagacaggcag 760 rno-miR-216 uaaucucagcuggcaacugug 761 rno-miR-217 uacugcaucaggaacugacuggau 762 rno-miR-218 uugugcuugaucuaaccaugu 763 rno-miR-219 ugauuguccaaacgcaauucu 764 rno-miR-221 agcuacauugucugcuggguuuc 765 rno-miR-222 agcuacaucuggcuacugggucuc 766 rno-miR-223 ugucaguuugucaaauacccc 767 rno-miR-290 cucaaacuaugggggcacuuuuu 768 rno-miR-291-5p caucaaguggaggcccucucu 769 rno-miR-291-3p aaagugcuuccacuuugugugcc 770 rno-miR-292-5p acucaaacugggggcucuuuug 771 rno-miR-292-3p aagugccgccagguuuugagugu 772 rno-miR-296 agggcccccccucaauccugu 773 rno-miR-297 auguaugugugcauguaugcaug 774 rno-miR-298 ggcagaggagggcuguucuucc 775 rno-miR-299 ugguuuaccgucccacauacau 776 rno-miR-300 uaugcaagggcaagcucucuuc 777 rno-miR-320 aaaagcuggguugagagggcgaa 778 rno-miR-321 uaagccagggauuguggguuc 779

Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it will be appreciated that various changes and modifications may be made without departing from the teachings herein. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings herein. 

We claim:
 1. A linker probe comprising a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion is capable of base pairing with the 3′ end region of an miRNA target nucleic acid, and wherein the 3′ end of the linker probe is extensible.
 2. The linker probe according to claim 1, wherein the linker probe is extensible by a reverse transcriptase.
 3. The linker probe according to claim 1, wherein the stem comprises 12-16 base-pairs.
 4. The linker probe according to claim 3, wherein the stem comprises 14 base-pairs.
 5. The linker probe according to claim 1, wherein the 3′ target specific portion comprises 5-8 nucleotides.
 6. The linker probe according to claim 1, wherein the loop corresponds to a universal primer portion.
 7. The linker probe according to claim 1, wherein the loop comprises 14-18 nucleotides.
 8. The linker according to claim 7, wherein the loop comprises 16 nucleotides. 